Method and device for illuminating a sample in a microscope in points

Abstract

The invention relates to a method for punctiform illumination of a sample (1) in a microscope, more particularly a MINFLUX microscope, using illumination light, with the sample (1) being sequentially illuminated at the illumination points (3) of a predefined or predefinable illumination point pattern (2). The method is distinguished in that a lateral extent of the illumination point pattern (2) is smaller than the longest wavelength of the illumination light and in that the illumination points (3) are always illuminated exclusively with a time offset and in that a distinct individual light source (4) of a plurality thereof is assigned to each illumination point (3) of the 10 illumination point pattern (2) and each illumination point (3) is illuminated by the focus of an illumination light bundle (5) of the individual light source (4) assigned thereto.

Claims

1. An apparatus for punctiform illumination of a sample (1) in a microscope, wherein an illumination point pattern (2) is predefinable, at the illumination points (3) of which the sample (1) is able to be sequentially illuminated, wherein the lateral extent of the illumination pattern (2) is smaller than the longest wavelength of the illumination light, and in that a distinct individual light source (4) of a plurality thereof is assigned to each illumination point (3) of the illumination point pattern (2), wherein each of the individual light sources (4) emits an illumination light bundle (5) which is focused on the illumination point (3) assigned thereto.

2. The apparatus as claimed in claim 1 being a MINFLUX microscope.

3. The apparatus as claimed in claim 1 having a control apparatus which controls the illumination process in such a way that the individual illumination points (3) of the illumination point pattern (2) are always exclusively illuminated with a time offset.

4. The apparatus as claimed in claim 2 having a control apparatus which controls the illumination process in such a way that the individual illumination points (3) of the illumination point pattern (2) are always exclusively illuminated with a time offset.

5. The apparatus as claimed in claim 1, wherein the lateral extent of the illumination pattern (2) is smaller than half the longest wavelength of the illumination light.

6. The apparatus as claimed in claim 2, wherein the lateral extent of the illumination pattern (2) is smaller than half the longest wavelength of the illumination light.

7. The apparatus as claimed in claim 1, wherein an imaging optical unit (6) is present, the latter imaging the illumination light bundle (5) into the illumination points (3).

8. The apparatus as claimed in claim 2, wherein an imaging optical unit (6) is present, the latter imaging the illumination light bundle (5) into the illumination points (3).

9. The apparatus as claimed in claim 1, further comprising a phase modulator (17).

10. The apparatus as claimed in claim 1, further comprising a zoom optical unit.

11. The apparatus as claimed in claim 1, wherein the illumination light bundle (5) focused on the illumination point (3) has an intensity minimum.

12. The apparatus as claimed in claim 2, wherein the illumination light bundle (5) focused on the illumination point (3) has an intensity minimum.

13. The apparatus as claimed in claim 1, wherein the focus on the illumination point (3) of the illumination light bundle (5) is in form of a 3D-Doughnut and/or the focus of the illumination light bundle (5) has a three-dimensional structure with a central intensity minimum, which is both a lateral and an axial intensity minimum.

14. The apparatus as claimed in claim 2, wherein the focus on the illumination point (3) of the illumination light bundle (5) is in form of a 3D-Doughnut and/or the focus of the illumination light bundle (5) has a three-dimensional structure with a central intensity minimum, which is both a lateral and an axial intensity minimum.

15. The apparatus as claimed in claim 7, wherein the distance of an illumination point (3) from each of its directly adjacent illumination points (3) is less than λ/NA in each case, where λ is the longest wavelength of the illumination light bundle (5) and NA is the numerical aperture of the imaging optical unit (6).

16. The apparatus as claimed in claim 8, wherein the distance of an illumination point (3) from each of its directly adjacent illumination points (3) is less than λ/NA in each case, where λ is the longest wavelength of the illumination light bundle (5) and NA is the numerical aperture of the imaging optical unit (6).

17. The apparatus as claimed in claim 1, wherein i). the individual light sources (4) each emit illumination light of a plurality of wavelengths, and/or in that ii). the individual light sources (4) emit illumination light of different wavelengths relative to one another.

18. The apparatus as claimed in claim 1, wherein the illumination points (3) are additionally able to be illuminated by the focus of a further light.

19. The apparatus as claimed in claim 17, wherein the illumination points (3) are additionally able to be illuminated by the focus of a further light bundle in each case, wherein iii). the further light bundles are emitted by the individual light sources (4) or wherein iv). the further light bundles are emitted by further individual light sources (4), wherein a distinct further individual light source (4) of a plurality thereof is in each case assigned to different illumination points (3) of the illumination point pattern (2).

20. The apparatus as claimed in claim 19, wherein i) the further light bundle has a different wavelength to the illumination light bundle (5) illuminating the same illumination point and/or in that ii) the further light bundle has a different wavelength to the illumination light bundle (5) illuminating the same illumination point and/or in that iii) the focus of the further light bundle has a complementary intensity distribution to the focus of the illumination light bundle (5) illuminating the same illumination point and/or in that iv) the focus of the illumination light bundle (5) has an intensity minimum and in that the further light bundle has a focus with an intensity maximum located where the intensity minimum of the focus of the illumination light bundle (5) is located.

21. The apparatus as claimed in claim 18, wherein i) the further light bundle has a different wavelength to the illumination light bundle (5) illuminating the same illumination point and/or in that ii) the further light bundle has a different wavelength to the illumination light bundle (5) illuminating the same illumination point and/or in that iii) the focus of the further light bundle has a complementary intensity distribution to the focus of the illumination light bundle (5) illuminating the same illumination point and/or in that iv) the focus of the illumination light bundle (5) has an intensity minimum and in that the further light bundle has a focus with an intensity maximum located where the intensity minimum of the focus of the illumination light bundle (5) is located.

22. The apparatus as claimed in claim 1, wherein the illumination duration at each of the illumination points (3) of the illumination point pattern (2) is adjustable and/or in that the illumination duration at each of the illumination points (3) of the illumination point pattern (2) is no more than 50 μs.

23. The apparatus as claimed in claim 1, wherein the illumination intensity of the illumination light bundles (5) and/or the intensity distribution in the focus of the illumination light bundles (5) and/or the further illumination light bundles (5) is adjustable individually or together.

24. The apparatus as claimed claim 1, wherein a distinct light intensity modulator (11) is arranged in the bundle path of each illumination light bundle (5) and/or further light bundle (5).

25. The apparatus as claimed in claim 24, wherein the light intensity modulators (11) are embodied as acousto-optic light intensity modulators (11) or as electro-optic light intensity modulators (11) or as liquid crystal modulators.

26. The apparatus as claimed in claim 1, wherein each individual light source (4) is respectively formed by a primary light source (7), in particular formed by a laser or semiconductor laser.

27. The apparatus as claimed in claim 1, wherein each illumination light bundle (5) is guided by an optical fiber (13) and/or in that different illumination light bundles (5) are always guided by different optical fibers (13).

28. The apparatus as claimed in claim 1, wherein the individual light sources (4) are formed by spatially splitting the light of a primary light source (7) into the illumination light bundles (5).

29. The apparatus as claimed in claim 28, wherein the primary light source (7) emits pulsed light (8).

30. The apparatus as claimed in claim 28, wherein the spatial split is realized by coupling different light components of the illumination light of the primary light source (7) into different optical fibers (13).

31. The apparatus as claimed in claim 18, wherein i) the optical path lengths of the bundle paths of the illumination light bundles (5) and/or of the further light bundles have different lengths, or in that ii) optical path lengths of the bundle paths of the illumination light bundles (5) are adjustable together or individually, or in that iii) the optical path lengths of the bundle paths of the illumination light bundles (5) and/or of the further light bundles have different lengths and the difference in the optical path lengths causes a temporal retardation of successive illumination light pulses of different illumination light bundles (5) of at least 3 ns.

32. The apparatus as claimed in claim 27, wherein i) the optical fibers (13) are combined to form an optical fiber bundle (14), or in that ii) the optical fibers (13) are combined to form an optical fiber bundle (14) and are arranged relative to one another in a Cartesian or hexagonal grid.

33. The apparatus as claimed in claim 27, wherein the optical fibers (13) are embodied as single mode fibers and/or as polarization-maintaining fibers.

34. The apparatus as claimed in claim 7, wherein an imaging unit (6) is present which images the illumination light bundles in form of a predefined illumination point source pattern, the predefined illumination point source pattern is imaged identically in the sample representing the illumination points (3) in the sample.

35. The apparatus as claimed in claim 27, wherein an imaging optical unit (6) is present, the latter imaging the decoupling ends (55) of the optical fibers (13) into the illumination points (3).

36. The apparatus as claimed in claim 27, wherein the optical fibers (13) have different lengths.

37. The apparatus as claimed in claim 27, wherein the decoupling ends (55) of at least two optical fibers (13) are arranged in different optical planes.

38. The apparatus as claimed in claim 28, wherein the spatial split is realized by illuminating a plurality of mirrors of a micromirror array with the light (8) of the primary light source (7).

39. The apparatus as claimed in claim 38, wherein the primary light source (7) emits continuous light (8).

40. The apparatus as claimed in claim 9, wherein a micromirror device (39) is arranged in the light (8) having a multitude of illumination light bundles (42) upstream of the phase modulator (17), wherein to each light bundle (42) a mirror or a group of mirrors of the micromirror device (39) is assigned.

41. The apparatus as claimed in claim 38, wherein the micromirror device (39) is a digital micromirror device (DMD).

42. The apparatus as claimed in claim 40, wherein the micromirror device (39) is a digital micromirror device (DMD).

43. The apparatus as claimed in claim 40, wherein a microlens array (40) is present after the micromirror device (39), wherein each microlens of the microlens device (40) is assigned to a light bundle mirrored or light point source from a single mirror or a group of mirrors of the micromirror device (39).

44. The apparatus as claimed in claim 19, wherein an adjustable bundle deflection apparatus is present in the bundle path of the illumination light bundles (5) and/or the further light bundles, the foci of the illumination light bundles (5) and/or of the further light bundles for illuminating the illumination point pattern (2) being positionable relative to the sample (1) before and/or during an illumination procedure by means of said bundle deflection apparatus.

Description

[0071] The subject matter of the invention is illustrated in an exemplary and schematic manner in the drawing and will be described hereunder by means of the figures, wherein identical elements or elements of equivalent function are in most instances provided with the same reference signs even in different exemplary embodiments. In detail:

[0072] FIG. 1 shows a first exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample,

[0073] FIG. 2 shows a detailed view of a second exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample,

[0074] FIGS. 3 and 4 show detailed views of a third exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample,

[0075] FIG. 5 shows a detailed view of a fourth exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample,

[0076] FIG. 6 shows an exemplary embodiment in relation to a possible arrangement of the optical fibers 13 within an optical fiber bundle 14,

[0077] FIG. 7 shows a detailed illustration of a fifth exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample,

[0078] FIG. 8 shows a detailed view of an exemplary embodiment of an apparatus according to the present invention detailing the beam pattern of the illumination light bundles further according to the invention which contains an apparatus according to the invention for punctiform illumination of a sample,

[0079] FIG. 9 shows a detailed view of a exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample,

[0080] FIG. 10 shows a detailed view of a further exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample, and

[0081] FIG. 11 shows a detailed view of another exemplary embodiment of an apparatus for punctiform illumination of a sample.

[0082] FIG. 1 schematically shows a first exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample 1 in a microscope, more particularly a MINFLUX microscope, wherein an illumination point pattern 2 is able to be defined, at the illumination points 3 of which the sample 1 is able to be repeatedly illuminated in sequence. A distinct individual light source 4 of a plurality thereof is assigned to each illumination point 3 of the illumination point pattern 2, with each of the individual light sources 4 emitting an illumination light bundle 5 which is focused on the illumination point 3, which is assigned thereto, by means of an imaging optical unit 6 which, in particular, may contain a microscope objective 24 (not illustrated in this figure).

[0083] Each of the individual light sources 4 is formed as a primary light source 7, which may be embodied as a laser, for example.

[0084] FIG. 2 shows a detailed view of a second exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample 1. In the second exemplary embodiment, the individual light sources 4 are formed by spatially splitting the light 8 from a single primary light source 7. Beam splitters 9 connected in succession in cascaded form, which may be embodied as neutral density or polarization beam splitters, in particular, serve to split the light 8 of the primary light source 7. It is also possible for the beam splitters 9 to be embodied as dichroic beam splitters in order to impinge different illumination points 3 with illumination light of different wavelengths.

[0085] An SLM (spatial light modulator) 10 is arranged in the beam path of each of the illumination light bundles 5 as a constituent part of an imaging optical unit 6 (otherwise not illustrated in any more detail) which images the illumination light bundles 5 into the illumination points 3. By way of example, the SLM 10 can serve to realize a certain shape of the focus of the illumination light bundles 5 at the location of the illumination points 3.

[0086] Moreover, a light intensity modulator 11 is situated in the beam path of each of the illumination light bundles 5. By means of the light intensity modulators 11, it is possible to set and/or temporally modulate the luminous power of each individual illumination light bundle 5. By way of example, it is possible to repeatedly activate and deactivate the individual light sources 4 in a predefined or predefinable sequence by means of the light intensity modulators 11 in order to sequentially (temporally successively) illuminate the illumination points 3. By way of example, it is also possible that the individual light sources 4 are respectively activated and deactivated in a random sequence by way of a corresponding control of the light intensity modulators 11 in order to illuminate the illumination points 3 sequentially in random fashion.

[0087] Alternatively, it is also possible for the single primary light source 7 to be embodied as a pulsed light source, for example as a pulsed laser, such that the luminous power of the pulsed illumination light bundles 5 of each of the individual light sources 4 can be set on an individual basis with the aid of the light intensity modulators 11. In such an embodiment, the optical path lengths of the individual illumination light bundles 5 are preferably different such that the illumination light pulses of the individual illumination light bundles 5 take different lengths of time to arrive at the associated illumination points and the illumination of the illumination points 3 by the illumination light pulses is consequently implemented in sequence. Preferably, the apparatus is embodied in such a way that the optical path lengths of the individual illumination light bundles 5 are adjustable together or individually. By way of example, adjustable optical retardation paths may be present to this end.

[0088] FIG. 3 shows a detailed view of a third exemplary embodiment of an apparatus according to the invention. In this exemplary embodiment, the illumination light beams 5 are each coupled into an optical fiber 13 by means of an input coupling optical unit 12 after they have respectively passed a light intensity modulator 11. The optical fibers 13 are combined to form a fiber bundle 14. The illumination light bundles 5 emerging from the optical fibers 13 of the optical fiber bundle 14 are collimated by means of a collimator 53 and deflected in such a way that they intersect in a plane, the Fourier plane 16, with respect to the focal plane (not illustrated) in which the illumination points are arranged. By way of example, it is advantageously possible to arrange a phase modulator 17 in this Fourier plane 16 in order, for example, to influence the shape of the focus of each of the illumination light bundles 5 at the location of the illumination points 3.

[0089] FIG. 4 illustrates how a distance Δy from the optical axis 21 is converted into an angle of incidence α in respect of the Fourier plane 16.

[0090] FIG. 5 shows a detailed view of a fourth exemplary embodiment of an apparatus according to the invention. In this exemplary embodiment, the decoupling ends 55 which are arranged in an image plane 38, that corresponds to the focal plane 54, i.e. is conjugate to it, are imaged on the illumination points 3 (not illustrated in this figure) by means of an imaging optical unit 6. The illumination light bundles 5 emerging from the decoupling ends 55 of the optical fibers 13 of the optical fiber bundle 14 are collimated by means of a collimator 53 such that they intersect in the Fourier plane 16 of the focal plane 54. A phase modulator 17, which is embodied as vortex phase plate in exemplary fashion, is arranged in this Fourier plane 16. Alternatively, the phase modulator may be a device allowing the generation of three-dimensional structures including three-dimensional doughnuts as described. Namely, these three-dimensional structures have a specific distribution of intensity with a minimal intensity, ideally zero intensity. In particular, the phase modulator may generate a structure which has all three spatial directions differences in intensity. For example, the phase modulator may be spatial light modulator (SLM), said SLM allows the generation of three-dimensional as well as two-dimensional doughnuts or other structures, including a so-called bottle-beam etc.

[0091] This exemplary phase modulator 17 is also illustrated separately in FIG. 5. In general, the phase modulator 17 is distinguished in that it impresses on a light beam passing therethrough a different phase retardation depending on location. The phase modulator 17 can be embodied in such a way that a donut-shaped focus of the illumination light bundles 5 is attained in the sample 1. Such a focus has an intensity minimum, more particularly an intensity zero, on the optical axis 21. The phase modulator 17 can also have such an embodiment that a focus of the illumination light bundles 5 with a three-dimensional structure is attained in the sample 1, an intensity minimum of said focus being surrounded both laterally, for example by way of a ring-shaped intensity distribution, and axially, for example by two dome-shaped intensity distributions. Such a focus may be referred to as 3D donut.

[0092] In FIG. 8, see below, an embodiment detailing further the presence of a micromirror array 39 and a microlens array 40 in the beam path is shown. Namely, additional components of the optical imaging unit located upstream of the collimator 53 are present.

[0093] In an intermediate image plane 18, the apparatus comprises an adjustable beam deflection device 19 which comprises a first adjustable pair of mirrors 20 and a second adjustable pair of mirrors not illustrated here. The pair of mirrors 20 and the pair of mirrors not illustrated pivot the beam about mutually perpendicular axes such that the foci of the illumination light bundles 5 can be positioned in the x-direction and in the y-direction within the sample. A scanning lens 22 is disposed upstream of the adjustable beam deflection device 19 and a tube lens 23 is disposed downstream thereof. The imaging optical unit 6 contains a microscope objective 24 which focuses the illumination light bundles 5 on the illumination points 3 of the illumination point pattern 2 within the sample 1.

[0094] The illumination light bundles 5 emanating from the optical fiber bundle 14 can be generated by splitting the light 8 of a single primary light source 7, as illustrated in FIG. 2, for example. It is also possible that the illumination light bundles 5 originate from individual light sources 4 which are all embodied as primary light sources 7, as is illustrated in FIG. 1, for example.

[0095] Alternatively, it is also possible for the individual light sources 4 to be formed by spatially splitting the light 8 of a primary light source 7 into the illumination light bundles 5, the spatial splitting being realized by coupling different light components of the light 8 of the primary light source 7 into the optical fibers 13. To this end, the input end of the optical fiber bundle 14 can be, for example, illuminated over a large area by the light 8 of the primary light source 7 such that, ultimately, the decoupling ends 55 of the optical fibers 13 act as the individual light sources 4. Alternatively, provision can be made in view of a particularly good input coupling efficiency for different light components of the light 8 of the primary light source 7 to be focused into the individual optical fibers 13 of the optical fiber bundle 14 in targeted fashion. By way of example, a microlens array adapted to the spatial conditions of the optical fiber bundle 14 can advantageously be used to this end. By way of example, it is also possible to couple each light component into respectively one of the optical fibers 13 using a separate optical unit.

[0096] FIG. 6 shows an exemplary embodiment in relation to a possible arrangement of the optical fibers 13 within an optical fiber bundle 14. The spatial arrangement of the decoupling ends 55 of the optical fibers 13 defines the relative spatial arrangement of the illumination points 3, indicated in the lower part of the figure by + signs, apart from scaling by the respective imaging scale. The decoupling ends 55 of four optical fibers 13, which act as four individual light sources 4, are arranged relative to one another in a y-shaped pattern, with the optical fiber bundle 14 also containing empty positions 25.

[0097] A first optical fiber 26 of the optical fibers 13 is assigned to a first illumination point 27 within the sample. A second optical fiber 28 of the optical fibers 13 is assigned to a second illumination point 29 within the sample. A third optical fiber 44 of the optical fibers 13 is assigned to a third illumination point 30 within the sample 1. A fourth optical fiber 31 of the optical fibers 13 is assigned to a fourth illumination point 32 within the sample.

[0098] The illumination light bundles 5 emerging from the optical fibers 13 are input coupled into the optical fibers 13 in pulsed fashion such that initially a light pulse emerges from the first optical fiber 26 such that the first illumination point 27 is illuminated with the first focus 33, which is configured as a donut-shaped focus. Subsequently, a light pulse emerges from the second optical fiber 28 such that the second illumination point 29 is illuminated with a corresponding donut-shaped second focus 34. Thereafter, a light pulse emerges from the third optical fiber 44 such that the third illumination point 30 is illuminated with a corresponding donut-shaped third focus 35. Finally, a light pulse emerges from the fourth optical fiber 31 such that the fourth illumination point 32 is illuminated with a donut-shaped fourth focus 36. Subsequently, the sequence is repeated again starting with the illumination of the first illumination point 27.

[0099] FIG. 7 shows a detailed illustration of a fifth exemplary embodiment of an apparatus according to the invention. In this exemplary embodiment, the pulsed light 8 of a primary light source 7, which may be embodied as a pulsed laser, for example, is split spatially into individual illumination light bundles 5 by means of a plurality of beam splitters 9. The illumination light bundles 5 are each coupled into an optical fiber 13 by means of an input coupling optical unit 37. The optical fibers 13 are embodied with different lengths such that the individual light pulses of the illumination light bundles 5 emerge temporally in succession from the exit end 15 of the optical fiber bundle 14 formed by combining the optical fibers 13. This facilitates a sequential and repeated illumination of the illumination points 3 of an illumination point pattern 2 within a sample 1, in each case with the illumination light of an individual light source 4.

[0100] FIG. 8 shows a cut-out of right side of the beam path shown in FIG. 3. Namely, a multitude of illumination light bundles 42 emanating from the light 8 of the primary light source 7 are reflected by a micromirror array 39 reflecting the single illumination light bundle 42. Before being reflected at the micromirror array the multitude of illumination light bundles 42 can form a contiguous collimated light beam. Depending on the position of each of the mirrors, deflection of the light bundle occurs. As shown, three possible positions of the mirror, either the single mirror or as a group of mirrors, is possible, see dashed positions on the left and right of the mirror and the middle position. Depending on the position of the mirror, reflection occurs. If required, an absorber 41 is present absorbing deflected light not entering the microlens system 40. In a predetermined position of the mirror, the illuminating light bundles 5 representing the illumination point pattern (2) are directed into the lens system of a microarray of lenses, focusing each of the illuminating light bundles 42 into an image plane 38, each focus forming a illumination light point source, the illumination light bundles entering the collimator 53 for collimation in such a way that they intersect in an Fourier plane 16 with regard to the focal plane in the sample. Each of the mirrors of the micromirror array 39 may be controlled individually or as a group. In addition, the microlens array may contain lenses having the same focal length or having a different focal length. The micromirror array 39 is controlled in way, that at a given time point illumination light is reflected only to one single microlens and that the illumination points of the illumination point pattern (2) are illuminated sequentially.

[0101] FIG. 9 shows a detailed view of a sixth exemplary embodiment of an apparatus according to the invention. In this exemplary embodiment, the illumination light bundles 5 of the individual light sources 4 are coupled into the optical fibers 13 of an optical fiber bundle 14. The illumination light bundles 5 emerging from the optical fibers 13 are collimated by means of a collimator 53 and subsequently arrive at a zoom optical unit 47, the latter allowing the imaging scale, and hence the distance of the illuminated illumination points 3 relative from one another, to be adjusted. The zoom optical unit 47 contains a first lens 48, downstream of which there is an adjustable lens 49. Moreover, the zoom optical unit 47 contains a second lens 50 and a third lens 51. A phase modulator 17 for shaping the foci of the illumination light beams 5 is arranged between the adjustable lens 49 and the second lens 50. The zoom optical unit 47 is positioned immediately in front of an intermediate image plane 52. The illumination light bundles 5 emanating from the zoom optical unit 47 are focused on the illumination points 3 of the illumination point pattern 2 by means of a microscope objective 24 (not illustrated).

[0102] When localizing a fluorescence marker, it is possible, for example, to proceed in such a way that, initially, use is made of an illumination point pattern 2 in which the spacings of adjacent illumination points 3 are comparatively large. The illumination point pattern 2 is preferably chosen to be so large that a fluorescence marker to be localized is situated with certainty within the illumination point pattern 2. Here, the size of the illumination point pattern 2 to be chosen depends on the quality of the prior knowledge about the position of the fluorescence marker to be localized. Following a first localization iteration, in which illumination points 3 of the illumination point pattern 2 were each illuminated in time offset fashion by the focus of the illumination light of the illumination light bundle 5 of the individual light source 4 assigned thereto, there can be further localization iterations in an iteration process, said further localization iterations each being carried out with an illumination pattern 2 that has shorter distances between the illumination points 3 than the respective immediately preceding illumination pattern 2, wherein care is taken in each case (for example by a relative shift between the sample and illumination optical unit) for the fluorescence marker to be situated within the illumination pattern 2. This increases the accuracy of the localization from localization iteration to localization iteration.

[0103] A reduction in the distances between the illumination points 3 can be attained (both laterally and axially in view of three-dimensional illumination patterns 2) by means of the zoom optical unit 47. The zoom optical unit 47 can advantageously be embodied in such a way that the reduction in the axial distances of the illumination points 3 in a three-dimensional illumination pattern 2 is accompanied approximately quadratically with the reduction of the lateral distances.

[0104] An alternative embodiment of a device suitable for adjusting the size of the illumination point pattern 2 is shown in FIG. 11.

[0105] FIG. 10 shows a detailed view of a seventh exemplary embodiment of an apparatus according to the invention for punctiform illumination of a sample 1.

[0106] In this exemplary embodiment, the decoupling ends 55 of the optical fibers 13 of an optical fiber bundle 14 act as individual light sources 4. The illumination light bundles 5 (not illustrated in this figure) emerging from the decoupling ends 55 of the optical fibers 13 are imaged onto the illumination points 3 (not illustrated in this figure) of a three-dimensional illumination pattern 2 (not illustrated in this figure).

[0107] In the axial direction, the decoupling ends 55 of the optical fibers 13 are not all situated in the same plane. This takes account of the fact that the illumination points 3 of the three-dimensional illumination point pattern 2 are spaced apart not only laterally (xy-plane) but also in the direction of the optical axis 21 (z-direction). Some of the decoupling ends 55 of the optical fibers 13 protrude further forward in the axial direction than other decoupling ends 55, leading to the foci of the illumination light bundles 5 being arranged in different focal planes within the sample in order to be able to illuminate the illumination points 3 of the three-dimensional illumination pattern 5.

[0108] In this exemplary embodiment, there are three first decoupling ends 57 which end in a common axial plane and which are arranged at the corners of a first equilateral triangle. Moreover, there is a second decoupling end 58 centrally in the center of the optical fiber bundle 14 and it protrudes slightly further forward in the axial direction than the first decoupling ends 57. Moreover, there are three third decoupling ends 59 which end in a further common axial plane, which are likewise arranged at the corners of a second equilateral triangle and which protrude slightly further forward in the axial direction than the second decoupling end 58. The first equilateral triangle is rotated through 60 degrees in relation to the second equilateral triangle. In this way, it is possible to generate an illumination pattern 2 in which the illumination light bundles emanating from the first decoupling ends 57 and the third decoupling ends 59 illuminate the corners of an octahedron while the illumination light bundle emerging from the second decoupling end 58 illuminates the illumination point 3 located at the center of the octahedron.

[0109] Other illumination patterns are realizable by changing the number of optical fibers 13 in the optical fiber bundle 14 and/or by changing the arrangement of the decoupling ends 55 of the optical fiber bundles 14. By way of example, the illumination at the corners of a tetrahedron is possible, in particular to accurately localize a fluorescence marker in the interior of the tetrahedron. Very generally, it is largely possible to have any desired illumination pattern 2, in particular even those that have one or more illumination points 3 in the interior, in particular at the center, of a three-dimensional geometric figure.

[0110] FIG. 11 shows a further advantageous arrangement of decoupling ends 55 of the optical fibers 13 in an optical fiber bundle 14. The decoupling ends 55 are arranged at a center and on concentric rings around the center. Firstly, this arrangement allows the generation of more complex illumination point patterns 2, the illumination points 3 of which are illuminated in sequence, and secondly it however also facilitates a repetition of the sequential illumination of illumination points 3 while maintaining the shape of the illumination point pattern 2 but changing the size of the illumination point pattern 2. Such an arrangement can be used as the only device with which the spacing of the illumination points of an illumination point pattern from one another can be set or it can be used in addition to a further device with the aforementioned function, such as a zoom optical unit 47, for example.

[0111] The description in the description of the figures was provided in view of the MINFLUX method in particular. However, the arrangements shown develop corresponding advantages even if other methods are applied, for example the small field scanning method or methods combining MINFLUX triangulation and STED techniques, and are suitable for these. By way of example, in such methods it is possible to use one or more of the decoupling ends 55 for the illumination with excitation light while other decoupling ends 55 are used for sequential illumination with fluorescence prevention light, which then acts as illumination light.

LIST OF REFERENCE SIGNS

[0112] 1 Sample [0113] 2 Illumination point pattern [0114] 3 Illumination points [0115] 4 Individual light source [0116] 5 Illumination light beam [0117] 6 Imaging optical unit [0118] 7 Primary light source [0119] 8 Light [0120] 9 Beam splitter [0121] 10 SLM (spatial light modulator) [0122] 11 Light intensity modulator [0123] 12 Input coupling optical unit [0124] 13 Optical fiber [0125] 14 Optical fiber bundle [0126] 15 Exit end of the optical fiber bundle 14 [0127] 16 Fourier plane [0128] 17 Phase modulator [0129] 18 Intermediate image plane [0130] 19 Beam deflection device [0131] 20 Adjustable pair of mirrors [0132] 21 Optical axis [0133] 22 Scanning lens [0134] 23 Tube lens [0135] 24 Microscope objective [0136] 25 Empty position [0137] 26 Optical fiber [0138] 27 First illumination point [0139] 28 Second optical fiber [0140] 29 Second illumination point [0141] 30 Third illumination point [0142] 31 Fourth optical fiber [0143] 32 Fourth illumination point [0144] 33 First focus [0145] 34 Second focus [0146] 35 Third focus [0147] 36 Fourth focus [0148] 37 Input coupling optical unit [0149] 38 Image plane [0150] 39 Micromirror array [0151] 40 Microlens array [0152] 41 Absorber [0153] 42 illumination light bundle [0154] 47 Zoom optical unit [0155] 48 First lens [0156] 49 Adjustable lens [0157] 50 Second lens [0158] 51 Third lens [0159] 52 Intermediate image plane [0160] 53 Collimator [0161] 54 Focal plane [0162] 55 Decoupling ends of the optical fibers 13 [0163] 56 Detection optical fiber bundle [0164] 57 First decoupling end [0165] 58 Second decoupling end [0166] 59 Third decoupling end