Method for operating a light microscope with structured illumination and optic arrangement

Abstract

A method for operating a light microscope with structured illumination includes: providing illumination patterns by means of a structuring device which splits impinging light into at least three coherent beam parts which correspond to a −1., 0., and +1. diffraction orders of light; generating different phases of the illumination patterns by setting different phase values for the beam parts with phase shifters; and recording at least one microscope image for each of the illumination patterns and calculating a high resolution image from the microscope images. Phase shifters are provided not only for the beam parts of the −1. and +1. diffraction orders but also at least one phase shifter for the beam part of the 0. diffraction order. At least two different phase values Φ.sub.0 are set with the at least one phase shifter for the 0. diffraction order to provide a plurality of illumination patterns with different phases.

Claims

1. A method for operating a light microscope with structured illumination, comprising: providing illumination patterns by means of a structuring device which splits impinging light into at least three coherent beam parts which correspond to a −1., 0. and +1. diffraction orders of light; providing phase shifters not only for the beam parts of the −1. and +1. diffraction orders but also at least one phase shifter for the beam part of the 0. diffraction order; generating different phases of the illumination patterns by setting different phase values for the beam parts with phase shifters, comprising setting at least two different phase values Φ.sub.0 with the at least one phase shifter for the 0. diffraction order; and recording at least one microscope image for each of the illumination patterns and calculating a high resolution image from the recorded microscope images.

2. The method as defined in claim 1, further comprising: setting two different phase values Φ.sub.0 with the phase shifter for the 0. diffraction order, which phase values Φ.sub.0 differ from each other by π, and recording at least one microscope image for each set phase value Φ.sub.0 which are used to calculate the high resolution image.

3. The method as defined in claim 2, further comprising: setting a plurality of different phase values with the phase shifters of the −1. and +1. diffraction orders for each of the two phase values Φ.sub.0 of the phase shifter for the 0. diffraction order; and recording at least one microscope image for each set phase value, and calculating the high resolution image from these microscope images.

4. The method as defined in claim 1, further comprising: changing settings of the two phase shifters of the −1. and +1. diffraction orders in opposite directions, starting from an operating point, for providing the different illumination patterns.

5. The method as defined in claim 4, further comprising: changing settings of the two phase shifters of the −1. and +1. diffraction orders in opposite directions, starting from an operating point, such that the two set phase values of the −1. and +1. diffraction orders are changed by the same absolute value but with different +/− sign.

6. The method as defined in claim 1, further comprising: changing settings of the phase shifters of the −1. and +1. diffraction order in an interval which at most spans a phase shift of a first order modulation contrast of π, for providing the different illumination patterns; and additionally setting a change of the phase value Φ.sub.0 with the phase shifter for the 0. diffraction order, for providing phase shifts of a first order modulation contrast corresponding to a phase shift larger than π.

7. The method as defined in claim 1, further comprising: for providing the different illumination patterns, changing settings of the phase shifters of the −1. and +1. diffraction orders such that always
0π≤(Φ.sub.1−Φ.sub.−1)/2≤1π applies, wherein (Φ.sub.1−Φ.sub.−1)/2 indicates a phase value of the first order modulation contrast, Φ.sub.1 indicates the phase value of the 1. diffraction order, and Φ.sub.−1 indicates the phase value of the −1. diffraction order, wherein for providing a phase shift of the first order modulation contrast within an interval from 1π to 2π, changing the phase value Φ.sub.0 for the 0. diffraction order by π and still changing settings of the phase shifters of the −1. and +1. diffraction orders such that always
0π≤(Φ.sub.1−Φ.sub.−1)/2≤1π applies.

8. The method as defined in claim 1, wherein the structuring device comprises a waveguide chip which splits impinging light into at least three path sections leading to at least three exit ports, wherein the at least three exit ports and their respective path sections correspond to the −1., 0. and +1. diffraction orders of light.

9. The method as defined in claim 8, wherein the phase shifters are provided at the waveguide chip at the path sections of the −1., 0. and +1. diffraction orders.

10. The method as defined in claim 9, wherein the phase shifters are one of: thermal phase shifters, piezoelectric phase shifters, electro-optic phase shifters or acousto-optic phase shifters.

11. The method as defined in claim 10, wherein a maximal difference between settings of the phase shifters of the −1. and +1. diffraction orders, i.e., a maximal temperature difference between two thermal phase shifters, or a maximal voltage difference between settings of two piezoelectric or electro-optic phase shifters or a maximal frequency difference between settings of two acousto-optic phase shifters, is at most as large that (Φ.sub.1−Φ.sub.−1)/2≤1π still applies at the maximal difference, wherein Φ.sub.1−Φ.sub.−1 is the difference between the phase shifts of the −1. and +1. diffraction orders that are set for the maximal difference.

12. The method as defined in claim 1, further comprising: setting two different phase values Φ.sub.0 for the 0. diffraction order when recording the microscope images from which the high resolution image is calculated, which phase values Φ.sub.0 differ from each other by π and have absolute values such that a z-dependent modulation contrast of the illumination pattern has a maximum in a detection plane which is sharply imaged onto a detector, wherein z is the propagation direction of the light.

13. An optic arrangement for structured illumination in a light microscope, comprising[ ]: a structuring device configured to split impinging light in at least three coherent beam parts which correspond to a −1., 0. and +1. diffraction orders of light; a plurality of phase shifters configured to shift the phases of the beam parts for setting different phases of illumination patterns; and a control unit configured for: setting different phase shifts not only with the phase shifters of the −1. and +1. diffraction orders, but also setting at least two different phase values with the phase shifter or phase shifters belonging to the 0. diffraction order; and calculating the high resolution image from the thus recorded microscope images.

14. The optic arrangement as defined in claim 13, wherein the structuring device comprises a waveguide chip configured to split impinging light into at least three path sections leading to at least three exit ports, wherein the at least three exit ports and their respective path sections correspond to the −1., 0., and +1. diffraction orders of light.

15. A light microscope comprising an optic arrangement as defined in claim 13.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Further features of the invention are described in the following with reference to the attached schematic figures:

(2) FIG. 1 is a schematic illustration of the wave number vectors of three light beams which correspond to 0., −1. and +1. diffraction orders and which shall interfere with each other;

(3) FIG. 2 schematically shows an embodiment of a light microscope with an optic arrangement according to the invention;

(4) FIG. 3 schematically shows an embodiment of the optic arrangement of FIG. 2;

(5) FIG. 4 schematically shows an exemplary design of a part of the optic arrangement of FIG. 2 or 3; and

(6) FIG. 5 schematically shows the positions and polarization directions of light beams at or behind a waveguide chip of a light microscope of the invention.

(7) Similar components are referred to with the same reference signs throughout all figures.

DETAILED DESCRIPTION OF THE DRAWINGS

(8) FIG. 2 shows schematically an embodiment of a light microscope 1 according to the invention comprising an optic arrangement 95 of the invention.

(9) The microscope 1 comprises a light source 4 which emits light 5 that can be guided via an input port selection device 8 to different input ports of a waveguide chip 90 of the optic arrangement 95. The waveguide chip 90 or the combination of a waveguide chip 90 and input port selection device 8 constitute a structuring device 91 by means of which light exiting the waveguide chip 90 is suitable for structured illumination microscopy (SIM).

(10) The input port selection device 8 is here formed by a scanner 8 with one or a plurality of pivotable mirrors or other pivotable light deflecting elements. In the following description, however, the scanner 8 may also be replaced by other variable beam deflection devices which may, in particular, be based on the acousto-optic principle.

(11) The light source 4 may comprise a plurality of lasers as indicated in FIG. 2. Between the light source 4 and the scanner 8, optionally cascaded mirrors 6 with a plurality of partially transmissive mirrors may be used to combine the beam paths of all lasers onto a common beam path. Furthermore optionally an acousto-optic tunable filter (AOTF) 7 and lenses 23 or a beam expander 23 may be provided.

(12) Depending on the deflection direction of the scanner 8, different input ports of the waveguide chip 90 can be selected. FIG. 2 shows, as an optional design, a plurality of optical fibers 11.1, 11.2 and 11.3 which guide light from the scanner 8 to the different input ports of the waveguide chip 90.

(13) Each input port of the waveguide chip 90 leads to a plurality of exit ports which are arranged in a dot pattern. Light that enters through an input port thus exits the waveguide chip 90 in a dot pattern. A dot pattern consists of coherent light beam bundles which may interfere in a specimen plane 20 to form an illumination pattern. As each input port of the waveguide chip 90 connects to different exit ports, it is possible to switch between different dot patterns.

(14) The exit ports of the waveguide chip 90 may be arranged in a pupil plane. A dot pattern in the pupil plane thus leads to a structured intensity pattern, for example stripes, in the specimen plane 20.

(15) Structured light 15 exiting the waveguide chip 90 is guided to the specimen plane 20 via different optical components which may comprise an optical element 18, for example a tube lens or a zoom arrangement 18, and an objective 19 in addition to further components.

(16) As shown in FIG. 2, the microscope 1 may also comprise a movable deflector 27 arranged between the scanner 8 and the waveguide chip 90. Depending on a position of the bypassing the waveguide chip 90.

(17) The deflector 27 may comprise two reflective surfaces 9 and 16 which are rigidly connected with each other. The reflective surface 9 reflects light from scanner 8 to one of the optical fibers 11.1, 11.2, 11.3. The reflective surface 16 reflects light 15 from the exit ports of the waveguide chip 90 to the optical element 18 and the objective 19.

(18) Light coming from the specimen is detected with a detector or camera 28. For example, a (dichroic) beam splitter 24 may be used to direct light coming from the specimen to the detector/camera 28 (and not to the waveguide chip 90). Another detector 22 may be used for a laser scanning process in which the deflector 27 is arranged such that light 5 is not guided through the waveguide chip 90. Optionally, lenses and filters 26 may be used in front of each of the detector 22 and the camera 28.

(19) A control unit 21 may be configured to control the phase shifters of the waveguide chip 90 which is described in more detail further below.

(20) Turning to FIG. 3, the design of an exemplary waveguide chip 90 is further described. The waveguide chip 90 comprises a substrate 70, for example quartz glass. Within the substrate 70, paths are formed which have another index of refraction than the substrate 70. It is thus possible to guide light along these paths. The waveguide chip 90 comprises a plurality of input ports 31, 41, 51 which are each connected with one path, referred to in the following as light guide paths 32, 42, 52. Optical fibers 11.1, 11.2, 11.3 may optionally be used to guide light to the different input ports 31, 41, 51.

(21) Each light guide path 32, 42, 52 leads to a respective splitter 33, 43, 53 which splits the light guide path 32, 42, 52 into a plurality of parts, referred to as path sections 34-36, 44-46, 54-56. Each path section leads to a respective exit port 37-39, 47-49, 57-59 where light exits the waveguide chip 90.

(22) The depicted example comprises a first, second and third input ports 31, 41, 51. The exit ports 37 to 39 connected with the first input port 31 form a first dot pattern. Analogously, the exit ports 47 to 49 of the second input port 41 and the exit ports 57 to 59 of the third input port 51 form a second and a third dot pattern, respectively.

(23) The waveguide chip 90 defines a plane P which may be arranged in or parallel to a pupil plane. In other words, the (main) directions of the light guide paths 32, 42, 52 span a plane P which is parallel to the pupil plane and may be arranged in the region of the pupil plane or in the pupil plane. All exit ports 37-39, 47-49, 57-59 are thus arranged in the region of or in the pupil plane.

(24) A dot pattern in the pupil plane spatially corresponds to the beam bundles of different diffraction orders of a grating which is conventionally arranged in an intermediate image plane. The diffraction orders comprise in particular a zeroth diffraction order which is a central beam part, and a −1. diffraction order and a +1. diffraction order which may have a common distance to the 0. diffraction order. In a pupil plane, these 0., −1. and +1. diffraction orders may form three dots, substantially along a line.

(25) A central path section 45 with its exit port 48 corresponds to the 0. diffraction order, wherein the exit port 48 may be located at or in the region of an optical axis of the light microscope. The two outer path sections 44, 46 with their respective exit ports 47, 49 correspond to the −1. and +1. diffraction orders. The light parts of the 0., −1. and +1. diffraction orders interfere in the specimen plane and form there an illumination pattern. For calculating a high resolution image, several specimen images with different illumination patterns are consecutively recorded. Differently oriented illumination patterns are here produced by consecutively illuminating the different input ports 31, 41, 51. Furthermore, for each illumination of one of the input ports, several microscope images are recorded which differ in the phase of the illumination pattern.

(26) To vary the phase of the illumination pattern, phase shifters 144, 145, 146 are arranged at the path sections 44, 45, 46 of the 0., −1. and +1. diffraction orders for varying optical path lengths in these path sections 44, 45, 46. The phase shifters 144, 145, 146 are integrated in the waveguide chip 90 and may for example be thermoelectric, piezoelectric, acousto-optic or electro-optic phase shifters. For example, a thermoelectric phase shifter changes the temperature of the respective path section and thus the optical path length. In this way, the phase shifter 145 changes a phase value Φ.sub.0 for light of the 0. diffraction order, the phase shifter 144 changes a phase value Φ.sub.−1 (also referred to as Φ.sub.2) for light of F the −1. diffraction order; and the phase shifter 146 adjusts a change of the phase value Φ.sub.1 for light of the +1. diffraction order. As an abbreviation, light of the +1. diffraction order” is used to refer to light that travels through the path section which has its exit port arranged such that (or from which exit port the light is guided such that), when interfering in the specimen plane, the light corresponds to a +1. diffraction order of a grating in an intermediate image plane (which grating is not used here).

(27) As explained in more detail above, the interfering beam parts of the 0., −1. and +1. diffraction orders form an illumination pattern with an intensity that oscillates with a first order modulation contrast and a second order modulation contrast. For varying the phase of the illumination pattern, it is necessary to vary the phases of the first and second order modulation contrasts. The first order modulation contrast is proportional to:

(28) $\cos \left[\frac{2\pi }{\lambda }\cos \left(\phi \right).Math.x+\frac{{\varphi }_1-{\varphi }_2}{2}\right].Math.\cos \left[\frac{2\pi }{\lambda }\left(1-\sin \left(\phi \right)\right).Math.z+\frac{2{\varphi }_0-{\varphi }_1-{\varphi }_2}{2}\right].$

(29) Its phase is varied in that the phase shifters 144-146 vary the phases Φ.sub.0, Φ.sub.1 and Φ.sub.2.

(30) Starting from an operating point, the phases Φ.sub.1 and Φ.sub.2 are changed in opposite directions. In case of a thermoelectric phase shifter, for example, it starts from an initial temperature which is higher than the ambient temperature and may n particular be equal for both sections 44, 46. Now one of the path sections 44, 46 is heated and the other of the path sections 44, 46 is heated less or is not heated such that its temperature drops. The temperatures of the two path sections 44, 46 are changed such that Φ.sub.1 and Φ.sub.2 are just changed by the same absolute value or have the same absolute value. Depending on whether or not there is a constant phase difference between Φ.sub.1 and Φ.sub.2 which is to be considered in the equations, the phase shifters 144, 146 may thus be controlled such that Φ.sub.1=−Φ.sub.2 or Φ.sub.1=−Φ.sub.2+const. is always true, wherein const. is a constant which is constant for the different phase settings with which the microscope images are recorded.

(31) Consider the above expression of the first order modulation contrast for the case that the phases of the +1. and −1. diffraction orders are varied according to Φ.sub.1=−Φ.sub.2, and Φ.sub.0 would not be varied (which is not part of the claimed matter); it is apparent that in this scenario:

(32) the phase expression

(33) 0$\frac{2{\varphi }_0-{\varphi }_1-{\varphi }_2}{2}$
in the second cosine term is constant for the different phase settings and thus does not contribute to the phase shifting,

(34) the phase expression

(35) $\frac{{\varphi }_1-{\varphi }_2}{2}$
in the first cosine term would have to be varied over an interval of 2π in order that the phase shifting covers a whole period.

(36) For

(37) $\frac{{\varphi }_1-{\varphi }_2}{2}$
to cover an interval of 2π, it is necessary to vary each of Φ.sub.1 and Φ.sub.2 by 2π, respectively, i.e., they must in particular have values within the interval 0π to 2π. This requires disadvantageously large temperature changes.

(38) In contrast, in some embodiments of the invention, Φ.sub.1 and Φ.sub.2 are each varied by only 1π (or less), and thus

(39) $\frac{{\varphi }_1-{\varphi }_2}{2}$
an interval of π. Nevertheless, variations over the whole phase region shall be possible. To this end, Φ.sub.0 may be changed by π. This changes the argument of the last cosine term in the above expression by π, thus changing the +/− sign of this cosine term. The change in the +/− sign corresponds to the mentioned variation of

(40) $\frac{{\varphi }_1-{\varphi }_2}{2}$
in the region of 1π to 2π. Advantageously, with the invention the phase can be shifted over the whole phase region of the intensity modulation, wherein Φ.sub.1 and Φ.sub.2 only need to be varied by rather small amounts and thus only moderate temperature changes are necessary at the path sections 44 and 46.

(41) The above descriptions of the path sections 44-46 and their respective phase shifters 144-146 similarly apply to the other path sections 34-36 and 54-56 with their respective phase shifters 134-136 and 154-156.

(42) Instead of the described phase shifters integrated in the waveguide chip, separate phase shifters may also be arranged in the beam path behind the waveguide chip. For example, transparent wobble plates may be used as phase shifters which provide different optical path lengths of transmitted light depending on their tilt angle.

(43) A high resolution image may now be calculated from a plurality of microscope images, wherein a respective microscope image is recorded for n different phase settings per illuminated input port of the waveguide chip. With the three input ports, 3n microscope images are thus recorded and taken into calculation to form one high resolution image. For all of these phase settings, the phase shifters may be controlled such that Φ.sub.1 and Φ.sub.2 are each set to several different values in an interval with an interval span of each 1π (or such that (Φ.sub.1−Φ.sub.2)/2 is set to several values in an interval spanning 1π), and additionally Φ.sub.0 is set to two values which differ from each other by π. Advantageously, the phase shifters only need to be adjusted over comparably small intervals.

(44) For a particularly good contrast in an interference pattern, the relative intensities of light from the exit ports 47 to 49 are relevant. The first order modulation contrast provides a z modulation and thus provides an axial resolution enhancement (i.e., in z direction/along the optical axis). The second order modulation contrast provides the lateral resolution enhancement. The splitter 43 may be configured such that the intensity in the central exit port 48 is lower than the intensities of the other exit ports 47, 49. For example, the intensity in the central exit port 48 may be between 2% and 25% of the light intensity reaching the splitter 43. The remaining light intensity is shared n equal parts among the two other exit ports 47, 49. These descriptions in turn apply similarly to the other path sections and exit ports.

(45) The groups of exit ports 37-39, 47-49 and 57-59 differ from each other in the arrangement of the resulting dot patterns. Each input port 31, 41, 51 connects with a central exit port 38, 48, 58 which are formed next to each other in a central region which may correspond to an optical axis of the microscope. Distances between the central exit ports 38, 48, 58 are smaller than distances to the other exit ports 37, 39, 47, 49, 57, 59 which are arranged on a circular band around this central region. As it is merely possible to form the central exit ports 38, 48, 58 next to each other and not at exactly the same position, the remaining exit ports 37, 39, 47, 49, 57, 59 may not be arranged exactly on a circle but rather on a circular band, wherein the exit ports 37 and 39 have the same distance to the exit port 38 and similarly the exit ports 47 and 49 have the same distance to the exit port 48 and the exit ports 57 and 59 have the same distance to the exit port 58.

(46) Different dot patterns that are rotated relative to each other correspond in the specimen plane to structured intensity patterns that are rotated relative to each other.

(47) In addition to the depicted path sections, light from an input port can also be distributed into several path sections. The exit ports of these path sections may be arranged to correspond to further diffraction orders, or may form other patterns.

(48) All exit ports of the waveguide chip 90 may be arranged in a common plane P 4 which is in or at a pupil plane. This is achieved in that light is coupled out of the waveguide chip 90 under an angle relative to the directions of the light guide paths 32, 42, 52. The angle may have any value that differs from 0. In particular, the angle to plane P may be approximately 90° or more generally between 20° and 90°.

(49) This is further described with reference to FIG. 4 which schematically shows details of the waveguide chip 90 of FIG. 3. FIG. 4 is a cross-sectional view of the waveguide chip 90 and thus this view is perpendicular to the view of FIG. 3.

(50) FIG. 4 shows the path section 34 which leads through substrate 70 to exit port 37. For deflecting and coupling-out light, a recess 72 is formed in the substrate 70, forming a surface 71 or interface 71 that directly borders path section 34. Light from the path section 34 is thus deflected at the interface 71, for example by total internal reflection, then crosses the waveguide chip 90 and exits the waveguide chip 90 at a side opposite to the recess 72 and opposite the interface 71. An angle of reflection at the interface 71 is approximately 90°. FIG. 4 shows in dashed lines a widened light beam, wherein a central dashed line 95 of the widened light beam indicates an exit direction 75 of the light. The exit direction is at an angle of (approximately) 90° to the plane of the waveguide chip. Light reflected by the interface 71 exits the waveguide chip 90 in an area which is referred to as an exit port 37. Such an area may be an interface between the substrate 70 and a surrounding medium, typically air.

(51) If the surface 71 is an interface between air and the substrate/the respective path section, then an evanescent light field penetrates the air. For typically used light wavelengths, this evanescent field may have a size of for example, approximately 100 nm. Due to interaction with air molecules, the evanescent field may cause damage to the surface 71. To avoid such problems, the surface 71 may be coated (for example with a metal or a dichroic layer) to reduce/avoid interaction of an evanescent field with air. Alternatively, the recess 72 may be provided with a cover and filled with a protective gas, for example Argon. The surface 71 then contacts the protective gas which does not interact with the evanescent field.

(52) Instead of using total internal reflection, it is also possible to provide a mirror at the surface 71.

(53) After the reflection at the interface 71, a light beam widens and thus exits the waveguide chip 90 with a larger cross-section compared with a case in which the light beam would leave the waveguide chip 90 at the interface 71 without previously crossing the substrate. The larger cross-section reduces a peak intensity over the cross-section. This reduced peak intensity ensures that no damages occur at the surface 37 at which the light exits the wave guide. Advantageously, no further elements (for example end caps as used at optical fibers) are necessary for coupling light out of the waveguide chip 90. Rather, light may simply exit the substrate 70 without further elements being required.

(54) Additional optical elements, for example a microlens or a half-wave plate, may be directly attached to the exit port (i.e., the area of the substrate where reflected light exits). Such an efficient arrangement and attachment of microlenses or other components would not be possible if light were to exit the waveguide chip at the side of the interface/mirror 71.

(55) The efficiency of each splitter depends on the polarization of incoming light. Preferably, the light is linearly polarized in a direction in which the light is split (as indicated with the arrows in FIG. 3). SIM requires, however, a polarization which is preferably perpendicular to this orientation. It is thus advantageous to rotate the polarization of the light after the splitter.

(56) This is further explained with reference to FIG. 5 which shows the three dot patterns in its left part. A first dot pattern comprises the light dots/spots 81, 82, 83 in a pupil plane corresponding to the exit ports 37 to 39. A second dot pattern comprises the light dots 84 to 86 which correspond to the exit ports 47 to 49. A third dot pattern finally comprises the light dots 87 to 89 corresponding to the exit ports 57 to 59. The polarization direction of each light spot 81 to 89 is indicated with an arrow.

(57) Two half-wave plates may be provided to rotate the polarization direction always by 90°. Such half-wave plates 25 are shown in FIG. 2 between the waveguide chip 90 and the specimen plane 20. The half-wave plates 25 may be directly arranged at the exit ports of the waveguide chip 90. In this way, smaller cross-sections of the plates 25 suffice. The polarization directions of the light dots/light spots 81-89 before and after the first half-wave plate are indicated with arrows in the central part of FIG. 5. This central part of FIG. 5 also shows the orientation of the fast axis 96 of the first half-wave plate. Note that only the angle between the axes of the two half-wave plates is relevant but not their absolute orientation. The r right part of FIG. 5 shows the polarization directions of the light spots 81-89 before and after the second half-wave plate. The fast axis 97 of the second half-wave plate is also indicated. As shown in the right part of FIG. 5, the polarization direction of each light spot is rotated by 90° and is then perpendicular to a line which connects the dots 81-83 of one dot pattern.

(58) Turning to FIG. 3, the surface of the substrate at each exit port 37 may also be provided with a structure acting as a half-wave plate. This replaces one of the two half-wave plates.

(59) The described optic arrangement comprising a waveguide chip allows fast switching between different illumination patterns that differ in their respective orientation and phase. The optic arrangement may be compact with only a small number of movable components. The advantageous phase variation at light guide paths corresponding to a 0. diffraction order allows to particularly efficiently provide illumination patterns with different phases. The exemplary embodiments shown in the figures may also be modified such that the structuring device is formed without the waveguide chip. For example, instead of the waveguide chip, splitters and optical fibers may be provided to produce the same dot patterns described with reference to the figures. Furthermore, a grating or phase modulators may be used instead of the waveguide chip to provide the coherent beam parts. For each beam part that is guided to the specimen plane, at least one respective phase shifter may be provided so that all beam parts can be phase-shifted independently from each other. Beam parts that are produced with a grating and for which there are no phase shifters may be filtered out before reaching the specimen plane.

(60) While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled n the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

LIST OF REFERENCE SIGNS

(61) 1 light microscope 4 light source 5 light 6 cascaded mirrors for combining the beam paths 7 AOTP 8 scanner 9, 16 reflective surfaces of the movable deflector 10 dichroic beam splitter 11.1, 11.2, 11.3 optical fibers 15 structured light exiting the waveguide chip 18 zoom arrangement 19 objective 20 specimen plane 21 control unit 22 detector 23 lenses 24 dichroic beam splitter 25 exit polarization unit with half-wave plates 26 filter 27 movable deflector 28 camera 31, 41, 51 input ports of the waveguide chip 32, 42, 52 light guide paths 33, 43, 53 splitters formed in the waveguide chip 34, 35, 36; 44, 45, 46; 54, 55, 56 path sections of the waveguide chip 37, 38, 39; 47, 48, 49; 57, 58, 59 exit ports of the waveguide chip 70 substrate of the waveguide chip 71 interface for deflecting light out of the waveguide chip 72 recess in the substrate of the waveguide chip 75 exit direction of light out of exit ports 81 to 89 light spots in a pupil plane 90 waveguide chip 91 structuring device 95 optic arrangement 96, 97 fast axes of the half-wave plates 134, 135, 136 phase shifters for the path sections 34, 35, 36 144, 145, 146 phase shifters for the path sections 44, 45, 46 154, 155, 156 phase shifters for the path sections 54, 55, 56 P plane defined by the waveguide chip.