Light microscope and method for providing structured illumination light

Abstract

A light microscope comprises: a structuring optical unit comprising a waveguide chip for providing a structured illumination; an input selection device for variably directing light to one of several inputs of the waveguide chip; the waveguide chip further comprising a light guide path following each of the inputs; each light guide path divides into several path divisions; and each path division leads to one output of the wave-guide chip. The outputs of the waveguide chip can be arranged at a pupil plane of the light microscope, and an exit direction of light from the outputs is transverse to a plane defined by the waveguide chip. A method for providing structured illumination light using the light microscope is also described.

Claims

1. A light microscope comprising: a structuring optical unit configured for providing a structured illumination from impinging light, the structuring optical unit comprising a waveguide chip with a plurality of inputs; an input selection device configured for variably directing light to one of the inputs; the waveguide chip further comprising a light guide path following each of the inputs wherein each light guide path divides into several path divisions; and each path division leads to one output of the waveguide chip; wherein an exit direction of light from the outputs is transverse to a plane defined by the waveguide chip, the path divisions are formed such that at least some of the path divisions which belong to different inputs intersect, and the intersecting path divisions intersect each other at an angle between 70° and 120°.

2. The light microscope as defined in claim 1, wherein the light guide paths and the path divisions extend in or parallel to a pupil plane of the light microscope.

3. The light microscope as defined in claim 1, wherein each output of the waveguide chip comprises an interface for deflecting light out of the waveguide chip by total internal reflection.

4. The light microscope as defined in claim 1, wherein each output of the waveguide chip comprises an interface for deflecting light out of the waveguide chip, wherein the interface comprises a mirror.

5. The light microscope as defined in claim 3, wherein each interface is at an angle between 20° and 70° to the plane of the waveguide chip.

6. The light microscope as defined in claim 3, wherein the interface is formed by a recess in the waveguide chip.

7. The light microscope as defined in claim 1, wherein the outputs of the waveguide chip that belong to the same input form a dot pattern, and the dot patterns are similar to each other but rotated relative to each other.

8. The light microscope as defined in claim 1, further comprising an input polarizing unit which is configured to polarize light such that its polarizing direction is in the plane of the waveguide chip when impinging on the inputs of the waveguide chip.

9. The light microscope as defined in claim 1, further comprising an output polarizing unit on which light from the outputs of the waveguide chip impinges, the output polarizing unit being configured to rotate a polarization direction of impinging light by 90°.

10. The light microscope as defined in claim 1, wherein each output of the waveguide chip comprises two mirrors arranged at a substrate of the waveguide chip, the mirrors being configured such that they rotate a polarization direction of impinging light by 90°.

11. The light microscope as defined in claim 1, wherein the waveguide chip comprises adjustable phase shifters at some or all of the path divisions, the phase shifters being configured for adjustably setting a phase shift of light in the respective path division.

12. The light microscope as defined in claim 1, wherein for providing a TIR illumination, the waveguide chip comprises one or more additional inputs which each connect to an additional light guide path leading to a respective TIR output of the waveguide chip, wherein the outputs of the waveguide chip define a geometric center, and wherein each TIR output is further away from the geometric center than any of the outputs of the waveguide chip.

13. The light microscope as defined in claim 1, further comprising: a zoom assembly arranged behind the outputs of the waveguide chip, a control unit designed to receive a control command indicating whether a structured illumination is desired or a total internal reflection illumination is desired, the control unit being designed to set a larger magnification with the zoom assembly if total internal reflection illumination is desired.

14. The light microscope as defined in claim 1, further comprising: optical elements arranged between the waveguide chip and a specimen plane and configured to: create a pupil plane at a location of the waveguide chip, wherein the outputs of the waveguide chip are arranged at the pupil plane, and to produce in the specimen plane an interference pattern of the light exiting through the outputs of the waveguide chip, for providing a structured illumination in the specimen plane.

15. The light microscope as defined in claim 1, wherein each light guide path is connected to a splitter formed in the waveguide chip which is configured to divide the light guide path into four parts, wherein three of said parts constitute three path divisions, and one of said parts leads light away such that light of that part does not illuminate a sample.

16. A method for providing structured illumination light in a light microscope, the method comprises the steps of: Guiding light from a light source to an input selection device; Variably directing light with the input selection device to one of a plurality of inputs of a waveguide chip which is configured to provide structured illumination from incoming light; the waveguide chip further comprising a light guide path following each of the inputs wherein each light guide path divides into several path divisions; and each path division leads to one output of the waveguide chip; wherein light exits the outputs of the waveguide chip in an exit direction which is transverse to a plane defined by the waveguide chip, the outputs of the waveguide chip that belong to the same input form a dot pattern, and the dot patterns are similar to each other but rotated relative to each other.

17. The method as defined in claim 16, wherein: the outputs of the waveguide chip are arranged at or in the region of a pupil plane of the light microscope.

18. The method as defined in claim 16, wherein the path divisions are formed such that at least some of the path divisions which belong to different inputs intersect; and the intersecting path divisions intersect each other at an angle between 70° and 120°.

19. The method as defined in claim 16, for providing a TIR illumination, the waveguide chip comprises one or more additional inputs which each connect to an additional light guide path leading to a respective TIR output of the waveguide chip, the outputs of the waveguide chip define a geometric center, and each TIR output is further away from the geometric center than any of the outputs of the waveguide chip.

20. A light microscope comprising: a structuring optical unit configured for providing a structured illumination from impinging light, the structuring optical unit comprising a waveguide chip with a plurality of inputs; an input selection device configured for variably directing light to one of the inputs; the waveguide chip further comprising a light guide path following each of the inputs wherein each light guide path divides into several path divisions; and each path division leads to one output of the waveguide chip; wherein an exit direction of light from the outputs is transverse to a plane defined by the waveguide chip, for providing a TIR illumination, the waveguide chip comprises one or more additional inputs which each connect to an additional light guide path leading to a respective TIR output of the waveguide chip, wherein the outputs of the waveguide chip define a geometric center, and wherein each TIR output is further away from the geometric center than any of the outputs of the waveguide chip.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Further features and characteristics of the invention will be described with respect to the attached figures.

(2) FIG. 1 schematically shows an embodiment of a light microscope with an optical assembly according to the invention;

(3) FIG. 2 schematically shows an embodiment of the optical assembly of FIG. 1;

(4) FIG. 3 schematically shows an exemplary design of a part of the optical assembly of FIG. 1 or 2;

(5) FIG. 4 schematically shows a further embodiment of an optical assembly of a light microscope according to the invention;

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

(7) FIG. 6 schematically shows another exemplary design of a part of the optical assembly of FIG. 1 or 2.

(8) Similar components are provided with identical reference signs throughout the figures.

DETAILED DESCRIPTION OF THE INVENTION

(9) FIG. 1 shows schematically an embodiment of an inventive light microscope 1 which comprises an optical assembly 95.

(10) The microscope 1 comprises a light source 4 which emits light 5 that can be guided via an input selection device 8 to different inputs of a waveguide chip 90 of the optical assembly 95. The waveguide chip 90 provides for structuring of the light such that light exiting the waveguide chip 90 is suitable for structured illumination microscopy (SIM), a superresolution microscopy technique. The input selection device 8 may also be regarded as part of the optical assembly 95.

(11) In the depicted example the input selection device 8 is formed as a scanner 8 with a rotatable mirror. However, in the following description the scanner 8 may also be replaced by other variants of an input selection device described elsewhere in this disclosure.

(12) The light source 4 may comprise, as indicated in FIG. 1, several lasers. Between the light source 4 and the scanner 8, a plurality of optical components is optionally provided: Following the light source 4, several partially transparent mirrors 6 may be used for combining the beam paths of all lasers on a common beam path. Furthermore, an acousto-optical tunable filter (AOTF) 7 and lenses 23 or a beam expander 23 may optionally be provided.

(13) The scanner 8 is configured to variably deflect impinging light 5 in different directions. To this end the scanner 8 may comprise one or more rotatable mirrors or other movable optical elements. These allow fast switching times. A fast scanner 8 may also be realized by varying a refraction index of a medium by acoustic waves transmitted through the medium.

(14) Light 5 deflected by the scanner 8 can be guided to the waveguide chip 90. Depending on the deflection direction of the scanner 8, different inputs of the waveguide chip 90 can be selected. FIG. 1 shows as an optional design several optical fibers 11.1, 11.2, and 11.3 which guide light from the scanner 8 to the different inputs of the waveguide chip 90.

(15) The design of the waveguide chip 90 is described in greater detail further below. Each input of the waveguide chip 90 leads to several outputs arranged in a spot pattern. Light entering one input thus exits the waveguide chip 90 in a spot pattern. The spot pattern consists of coherent light beam bundles. These are thus suitable to interfere in a specimen plane 20, creating an interference pattern used as structured illumination. As each input of the waveguide chip 90 is connected to different outputs, it is possible to choose between different spot patterns.

(16) As an important characteristic of the invention, the outputs of the waveguide chip 90 are arranged in a pupil plane. Hence, a spot pattern in the pupil plane leads to a structured pattern of, e.g., stripes in the specimen plane 20. Different spot patterns in the pupil plane lead to different structured patterns in the specimen plane, as required for structured illumination microscopy.

(17) Structured light 15 exiting the waveguide chip 90 is guided to the specimen plane 20 via several optical components, which may include an optical element 18 such as a tube lens or a zoom assembly 18, and an objective 19, amongst others.

(18) As shown in FIG. 1, the microscope 1 may also comprise a movable deflector 27 arranged between the scanner 8 and the optical waveguide chip 90. Depending on a position of the deflector 27, light is either directed to the waveguide chip 90 or is bypassed around the waveguide chip 90 directly to the specimen plane 20. In the position shown in FIG. 1, the movable deflector 27 is arranged such that it directs light 5 from the scanner 8 to the optical fibers 11.1, 11.2, 11.3 leading to the inputs of the waveguide chip 90. If the movable deflector 27 is moved out of the beam path, light 5 from the scanner 8 travels directly towards the optical element 18 or the objective 19.

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

(20) Light coming from the sample is detected with a detector or camera 28. For example, between the objective 19 and the deflector 27, or between the deflector 27 and the outputs of the waveguide chip 90, a (dichroic) beam splitter 24 may be used for guiding light coming from the specimen to the detector/camera 28 (and not to the waveguide chip 90). A further detector 22 may be provided for a laser scanning operation in which the deflector 27 is arranged such that light 5 does not travel via the waveguide chip 90. In front of the detector 22 and the camera 28, lenses and filters 26 may optionally be used, respectively. In a laser scanning operation, illumination light and specimen light may share a common beam path through the objective 19 and may be separated with a partially reflective mirror 10, e.g., a dichroic beam splitter 10 which transmits or reflects light depending on the wavelength.

(21) A control unit 21 may be configured to control the scanner 8, the detector 22, and/or movement of the movable deflector 27.

(22) The microscope 1 may further comprise thermo-electric or piezo-electric phase shifters which are preferably integrated in the waveguide chip 90. The phase shifters adjustably shift a phase of light, which is required for recording several images with structured illumination of the same orientation but different phase. For example, the waveguide chip 90 may comprise a thermo-electric unit or electrical heating elements next to the path divisions, for varying the temperature of the path divisions. In this way the phase of light running through the individual path divisions can be varied.

(23) Turning to FIG. 2, the design of an exemplary waveguide chip 90 is shown and will be further described.

(24) The waveguide chip 90 comprises a substrate 70, which may be fused silica, for example. Within the substrate 70 paths are formed which have a different refractive index compared with the substrate 70. It is thus possible to guide light along these paths. The waveguide chip 90 comprises several inputs 31, 41, 51, which are each connected to a respective path, in the following referred to as light guide paths 32, 42, 52. Optical fibers 11.1, 11.2, 11.3 may be used to direct light to the different inputs 31, 41, 51. Depending on a state of the scanner 8, it is thus possible to selectively illuminate one of the inputs 31, 41, 51. The optical fibers may be omitted if the scanner is arranged such that it directs light directly onto one of the inputs 31, 41, 51.

(25) 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 several parts, referred to as path divisions 34-36, 44-46, 54-56. Each path division leads to a respective output 37-39, 47-49, 57-59, where light exits the waveguide chip 90.

(26) In the depicted example, there is a first, second, and third input 31, 41, 51. The outputs 37-39 connected with the first input 31 form a first spot pattern; the outputs 47-49 connected with the second input 41 form a second spot pattern; and the outputs 57-59 connected with the third input 51 form a third spot pattern.

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

(28) A spot pattern in the pupil plane corresponds spatially to the beam bundles of different diffraction orders of a grating arranged in an intermediate image plane. In prior art setups, such a grating is used for providing structured illumination. Light diffracted at a grating forms several beam parts corresponding to different diffractions orders. The diffraction orders comprise in particular a zeroth diffraction order which is a central beam part, and a minus first and first diffraction orders. In a pupil plane these zeroth, minus first, and first diffraction orders may form three dots along one line. The outputs 37-39, 47-49, 57-59 are now arranged to produce such a spot pattern in the pupil plane. For easier understanding, reference is in the following made only to the outputs 37-39. A central output 38 is arranged at a center point which may be at or in the region of an optical axis of the light microscope. This central output 38 provides a light spot corresponding to a zeroth diffraction order of a grating in an intermediate image plane. The other two outputs 37, 39 are arranged opposite each other with respect to the central output 38, and correspond to the minus first and first diffraction orders, respectively.

(29) Light parts from these outputs 37-39 interfere in the specimen plane. For a particularly good contrast in such an interference pattern, the relative light intensities of the outputs 37-39 are important. Preferably the splitter 33 is designed such that the intensity in the central output 38 is lower than in the other outputs 37, 39. For example, the intensity in the central output 38 may be between 2% and 25%, or between 8% and 20% of the light intensity reaching the splitter 33. The remaining light intensity is distributed in equal parts to the two other outputs 37, 39.

(30) The description above to the outputs 37-39 and the components connected thereto also applies to the outputs 47-49, and 57-59.

(31) These groups of outputs 37-39, 47-49, and 57-59 differ in the arrangement of their spot patterns. Each spot pattern may be formed by spots in a line; however, the lines of different groups of outputs 37-39, 47-49, and 57-59 are rotated relative to each other. In other words, each input 31, 41, 51 is connected with a central output 38, 48, 58; these are formed next to each other in a central region corresponding to an optical axis of the microscope. The remaining outputs 37, 39, 47, 49, 57, 59 are arranged on a circular band around said central region. As the central outputs 38, 48, 58 are merely next to each other and cannot be at exactly the same position, the remaining outputs 37, 39, 47, 49, 57, 59 are preferably not arranged on an exact circle but rather a circular band in which the outputs 37 and 39 have the same distance to the output 38, and similarly the outputs 47 and 49 have the same distance to the output 48, and the outputs 57 and 59 have the same distance to the output 58.

(32) Different spot patterns that are rotated relative to each other correspond to structured patterns in the specimen plane that are rotated relative to each other, as required for structured illumination microscopy.

(33) A crucial feature of the waveguide chip 90 described above resides in that all outputs are arranged in a common plane P which is in or at a pupil plane. This is achieved by coupling-out light from the waveguide chip 90 at an angle relative to the directions of the light guide paths 32, 42, 52. The angle may in principle have any values that differ from zero; wherein it may be preferred that the angle to the plane P is approximately 90° (i.e., parallel to a normal of the plane P) or more generally between 20° and 90°.

(34) This is further explained with reference to FIG. 3, which schematically shows a detail of the waveguide chip 90 of FIG. 2. FIG. 2 is a cross-sectional view of the waveguide chip 90, and hence this view is perpendicular to the view of FIG. 2.

(35) FIG. 3 shows the substrate 70 in which the path division 34 is formed. The path division 34 leads to the output 37. For redirecting and coupling-out light, a recess 72 is provided in the substrate 70. The recess 72 may be regarded as part of the output 37 and forms a surface 71 or interface 71 which abuts the path division 34. Light from the path division 34 is thus deflected at the interface 71. In the depicted example, the surface 71 is at such an angle relative to the path division 34 that light is reflected within the waveguide chip 90 by total internal reflection. Reflected light thus passes through the waveguide chip 90 and exits the waveguide chip 90 at a side opposite the recess 72 and opposite the interface 71. A reflection angle at the interface 71 is approximately 90°. FIG. 3 shows in dashed lines a fanned light beam, wherein a central dotted line 75 of the fanned light beam indicates an exit direction 75 of light. The exit direction is at an angle of 90° to the plane of the waveguide chip; this plane extends along the path division 34 and into the paper plane of FIG. 3. A region at which light reflected from the interface 71 exits the waveguide chip 90 is referred to as an output 37. Such a region may simply be an interface between the substrate 70 and a surrounding medium, typically air.

(36) The surface 71 may also be referred to as a TIR (total internal reflection) micro mirror. If the surface 71 is an interface between air and the substrate/the respective path division, an evanescent light field penetrates the air. For typically used light wavelengths this evanescent field may be, for example, about 100 nanometers. Due to interaction with air molecules, the evanescent field may lead to a deterioration of the surface 71. To avoid such drawbacks, the surface 71 may be coated (e.g., with a metal or dichroidically coated) to avoid interaction of an evanescent field with air. Alternatively, the recess 72 may be provided with a cover and filled with a protective gas such as Argon. The surface 71 then contacts the protective gas which does not interact with the evanescent field.

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

(38) In other variants of the invention, the surface/interface 71 may be formed at another angle to cause an exit direction varying from 90°.

(39) The reflecting interface 71 is formed on one side of the substrate such that reflected light travels through the substrate before exiting the waveguide chip 90 on a side opposite the interface 71. After reflection at interface 71, a light beam widens and hence exits the waveguide chip 90 with a larger cross-section compared with a case in which the light beam would exit the waveguide chip 90 at the interface 71, without previous transversal of the substrate. The larger cross-section means a reduction in peak intensity over the cross-section. This reduced peak intensity ensures that no damage occurs at the surface where the light exits the waveguide. Advantageously, no further elements (such as end caps used at optical fibers) are necessary for coupling-out light from the waveguide chip. Instead, light may simply leave through the substrate without any further elements being necessary.

(40) The arrangement of the interface or mirror 71 such that reflected light is directed through the waveguide chip 90 has further advantages: Additional optical elements such as a microlens or a half-wave plate may be directly attached to the output (i.e., the location of the substrate where reflected light exits). Such a convenient arrangement and attachment of microlenses or other components would not be possible if light left the waveguide chip on the side of the interface/mirror 71.

(41) The other path divisions and other outputs may be formed similarly to the one shown in FIG. 3.

(42) While FIG. 2 shows an embodiment in which each splitter 33, 43, 53 splits incoming light intro three path divisions, other splitter design with additional path divisions are possible; the above description applies in those cases, wherein the additional outputs may be arranged on the same line as the three outputs described above, or alternatively in another pattern.

(43) A further embodiment of a waveguide chip 90 of an inventive light microscope is shown in FIG. 4. This waveguide chip 90 may comprise all components shown in FIG. 2, with additional features in the design of the splitters 33, 43, 53, and additional inputs 62, 72, 82 for TIR illumination, i.e., illumination suitable for examining a specimen by total internal reflection of light at a specimen surface.

(44) The features of the splitter design and the inputs for TIR illumination are independent from each other, and hence the optical assembly of FIG. 2 may additionally be provided with the splitter design of FIG. 4 and/or the features for the TIR illumination that will be described with reference to FIG. 4.

(45) The splitters 33, 43, 53 used in FIG. 4 split light into four path divisions 30, 34-36; 40, 44-46; and 50, 54-56, respectively. For easier understanding, the following description refers to splitter 33 and its path divisions 30, 34-36. However, the other splitters 43, 53 and their path divisions may be formed similarly.

(46) Only three of the four path divisions 34-36 lead to outputs as described with reference to FIG. 2. The respectively remaining path division 30 is not used to illuminate a sample. Instead, it may lead to an outer area further away from the optical axis and directs or scatters light to an absorber, e.g., a casing of the optical assembly. Each splitter 33 is built such that two of its path divisions 34, 36 receive an equal amount of light intensity which is higher than the light intensity in the other two path divisions 30, 35. These path divisions 30, 35 may again receive the same intensity. The path divisions 34, 36 may be centrally arranged in the splitter 33 whereas the other two path divisions 30, 35 are further outside in the splitter 33. Such a design with an unused path division (i.e., a path division not used to illuminate the sample) offers advantages in that the intensity distribution among the path divisions depends less on a wavelength of the light. This is further explained in the general description of this invention.

(47) The remaining path division 30 which is not used to illuminate a sample may be used for other purposes. It may guide light to a photodetector, e.g., by leading to an outer area of the pupil plane, and from there coupling-out light to a photodetector. Signals from the photodetector may be used to monitor and control the coupling-in of light at the inputs of the waveguide chip. For example, depending on a signal from the photodetector, an element connected with the input (e.g., a position of an optical fiber leading to this input) may be adjusted.

(48) Alternatively, the remaining path division 30 which is not used to illuminate a sample is connected to an interferometer with a phase shifter, e.g., a Mach-Zehnder-Interferometer. The phase shifter and preferably the interferometer are integrated in the waveguide chip. The phase shifter may be controlled identically to the other phase shifters in the waveguide chip, e.g., a common temperature change may be induced in all phase shifters. The interferometer measures the phase shift caused by this temperature change. Thus, a measurement value of the interferometer allows to correct a control of the phase shifters to achieve a desired phase shift. This may be implemented in a closed-loop (feedback) control.

(49) In further embodiments, a remaining path division 30 which is not used to illuminate a sample is connected with a light source. For example, an LED may be fixed on the waveguide chip at an end region of the path division 30. Light from the light source travels through path division 30 towards splitter 33. The light further propagates from splitter 33 through light guide path 32 and leaves the waveguide chip at input 31. After traveling through optical fiber 11.1, the light can be measured with, e.g., a photo detector, and can be used for beam adjustment or collimation adjustments.

(50) Other remaining path divisions 40, 50 not used for illuminating a sample may be similarly or alternatively provided with a light source and used as described above for path division 30.

(51) Instead of four path divisions, another even number may be used, wherein in each case one path division is not used to illuminate the sample.

(52) If one of the outer path divisions 35 receives a lower intensity, this path division should lead to a central portion corresponding to a zeroth diffraction order of a grating in an intermediate image plane. This may require intersecting or crossing another path division 36.

(53) Furthermore, path divisions 36, 46 of different splitters 33, 43 may intersect each other. Preferably, an angle of intersection (defined for the sections of the path divisions 36, 46 that run from the intersection to their respective outputs) is between 80 and 120°, or preferably equal or larger 90°. In this way crosstalk between the path divisions 36, 46 is low. An angle larger 90° means that a fraction of light from path division 36 that enters path division 46 is more likely to run backwards and not towards the output of path division 46; and hence does not or hardly affect the generated spot pattern. As described elsewhere in this disclosure, such intersections can be avoided with a 3D design of light guide paths or path divisions. In such a design the light guide paths or path divisions are not limited to a plane but also extend in a direction transverse to this plane.

(54) The waveguide chip 90 of FIG. 4 further comprises additional inputs 61-63 used for TIR illumination, which are thus also referred to as TIR inputs 61-63. Each TIR input 61-63 is connected to a respective TIR output 67-69 via a respective light guide path 64-66. In the pupil plane, the TIR outputs 67-69 are arranged further outside (i.e., further distanced from an optical axis) than the outputs 37-39, 47-49, 57-59 for structured illumination.

(55) A respective optical fiber may lead to each of the TIR inputs 61-63 such that the scanner 8 is able to select either one of the inputs 31, 41, 51, 61, 62, 63.

(56) While FIG. 4 shows an embodiment with three TIR inputs 61-63, this design may be generalized to one or more TIR inputs.

(57) The efficiency of each splitter depends on the polarization of incoming light. Preferably, the light is linearly polarized in a direction in which the splitting occurs (as indicated with arrows in FIGS. 2 and 4). However, SIM requires a polarization that is preferably perpendicular to this orientation. It is therefore preferred to rotate the polarization of light behind the splitters.

(58) This is further described with reference to FIG. 5, which shows in the left part the three spot patterns. A first spot pattern comprises light spots 81, 82, 83 in a pupil plane corresponding to the outputs 37-39. A second spot pattern comprises light spots 84-86 corresponding to the outputs 47-49. Finally, a third spot pattern comprises light spots 87-89 corresponding to the outputs 57-59.

(59) The polarization direction for each light spot 81-89 is indicated with an arrow.

(60) Two half-wave plates may be used for rotating the polarization direction in each case by 90°. Such half-wave plates 25 are shown in FIG. 1 between the waveguide chip 90 and the specimen plane 20. Preferably the half-wave plates 25 are arranged directly at the outputs of the waveguide chip 90. In this way, smaller cross-sections of the plates 25 suffice. The half-wave plates 25 are preferably achromatic. The polarizations of the light spots 81-89 before and after passing through the first half-wave plate are shown in the middle section of FIG. 5 with arrows. This middle section also indicates 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 important but not their absolute orientation. The right part of FIG. 5 shows the polarization directions of the light spots 81-89 before and after passing through the second half-wave plate. Indicated is also the fast axis 97 of the second half-wave plate. As shown in the right part of FIG. 5, the polarization direction for each light spot is rotated by 90° and is now perpendicular to a line connecting the spots 81-83 of the same spot pattern.

(61) One or both of the half-wave plates may also be substituted by other means that achieve a polarization rotation of 90°. Turning to FIG. 3, the surface of the substrate at each output 37 may be provided with or processed to have a structure acting as a half-wave plate, thus replacing one half-wave plate.

(62) Each path division may also lead to two consecutive mirrored interfaces before light exits the waveguide chip. Such an example is shown in FIG. 6, which schematically shows a perspective view of a part of a waveguide chip 90.

(63) A path division 34 leads to two consecutive mirrored surfaces 73 and 71. These are arranged in the form of a periscope, also referred to as a micro-periscope. The first mirrored surface 73 redirects light from path division 34 within the waveguide chip and/or within the plane of the waveguide chip to the second mirrored surface 71. The second mirrored surface 71 redirects light into the exit direction 75 out of the waveguide chip 90. A polarization direction of light in the path division 34 and before the mirrored surfaces 71, 73 is indicated with arrow 76. As shown, this polarization direction may be in the plane of the waveguide chip. Reflection at the surfaces 73, 71 affects the polarization such that a polarization direction 77 of light having left the waveguide chip 90 is rotated, preferably by 90°.

(64) In addition to the described light guide paths, a further light guide path may be provided which is not connected with one of the inputs 31, 41, 51 of the waveguide chip. The further light guide path may have an input coupled with a further light source, e.g., an LED connected to the waveguide chip at the input of the further light guide path. The further light guide path has an output located next to one of the inputs 31, 41, 51. For example, a distance between the output of the further light guide path and one of the inputs 31, 41, 51 may be smaller than five diameters of the (further) light guide path. In particular, a coupling-out direction of the output of the further light guide path may be parallel to a coupling-in direction of the inputs 31, 41, 51. This may be achieved in that the further light guide path is, in its end section to its output, parallel to the sections of the light guide paths 32, 42, 52 at their inputs 31, 41, 51. Light leaving the output of the further light guide path may be measured with a further light detector and/or used for beam adjustment or collimation adjustments.

(65) The inventive design offers an improved stability and accuracy as the outputs of the waveguide chip and hence the spot patterns in the pupil plane have a predefined arrangement, and no optical components are necessary to direct light from the waveguide chip to a pupil plane which would need to be aligned or may suffer misalignment over time. In particular no optical fibers are necessary for this purpose, and hence coherence problems due to different lengths of optical fibers are avoided. As a consequence, less expensive lasers can be used as light sources. A particularly high accuracy is achieved with a setup requiring a comparatively low number of components.

(66) A main advantage of the inventive light microscope resides in that many functions can be integrated in a single waveguide chip. This not only leads to space reductions but also avoids adjustment issues of individual movable components. For instance, optical fibers behind the waveguide chip become obsolete. Also phase shifters and/or polarization rotating means may be integrated in the waveguide chip; wherein the polarization rotating means may be formed by a mirror pairs in the shape of micro-periscopes or with a half-wave plate arranged at each of the outputs.

LIST OF REFERENCE SIGNS

(67) 1 light microscope 4 light source 5 light 6 mirrors for combining beam paths 7 AOTF 8 scanner 9, 16 reflecting 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 assembly 19 objective 20 specimen plane 21 control unit 22 detector 23 lenses 24 dichroic beam splitter 25 output polarizing unit with half-wave plates 26 filters 27 movable deflector 28 camera 30, 40, 50 path divisions not used for illuminating a sample 31, 41, 51 inputs of the waveguide chip 32, 42, 52 light guide path 33, 43, 53 splitter formed in the waveguide chip 34, 35, 36; 44, 45, 46; 54, 55, 56 path divisions of the waveguide chip 37, 38, 39, 47, 48, 49, 57, 58, 59 outputs of the waveguide chip 61, 62, 63 additional inputs 64, 65, 66 additional light guide path 67, 68, 69 TIR output 70 substrate of the waveguide chip 71 interface for deflecting light out of the waveguide chip 72 recess provided in the substrate of the waveguide chip 73 interface for deflecting light within the waveguide chip 75 exit direction of light from the outputs 76 polarization direction of light within the waveguide chip 77 polarization direction of light coupled-out of the waveguide chip 81-89 light spots in a pupil plane 90 waveguide chip 95 optical assembly 96, 97 fast axes of the half-wave plates P plane defined by the waveguide chip