DE102018110072A1 - Optical arrangement for structured illumination for a light microscope and method for this purpose - Google Patents

Abstract

An optical arrangement for structured illumination for a light microscope comprises: a polarization beam splitter (10) which splits incident light (2) polarization-dependent into reflection light (12A) and transmission light (12B); and a light structuring device (30) which imparts structurings different from each other to the transmission light (12B) and the reflection light (12A). The polarization beam splitter (10) is arranged so that it the transmission light (12B) and the reflection light (12A), which differ in their structuring and their polarization, reunited on a common beam path (55).

Description

The present invention relates in a first aspect to an optical arrangement for structured illumination for a light microscope according to the preamble of claim 1.

In a second aspect, the invention relates to a method for providing a structured illumination in a light microscope according to the preamble of claim 13.

Structured illuminations are used in SIM (Structured Illumination Microscopy) to increase the resolution. As a structuring, the light beam shaping or the manipulation of the point spread function (PSF engineering), as used for example for STED or RESOLFT microscopy method can be understood. The same applies to changes in the illumination light or detection light, as it is done for other microscopy methods, such as PALM / STORM or SOFI (Super Resolutions Optical Fluctuation Imaging).

In microscopy with structured illumination, a sample is illuminated with light that has been given a spatial structure. For example, the structuring in the sample plane may represent a grid or line grid, although other patterns with a light intensity varying over the cross section are also possible. Several sample images are taken in which differently structured illuminations are used. For example, three differently rotated illumination patterns can be used one after the other, with three different light phases also being used in succession, for example. Frequently, images are taken at five different light phases in order to achieve an increase in resolution in the z-direction. The thus recorded sample images with different structured illumination are then calculated into a high-resolution image.

A generic optical arrangement for structured illumination for a light microscope comprises a light structuring device which imparts structuring to incident light. In addition, the optical arrangement comprises a polarization beam splitter, which is arranged so that incident light is polarization-dependent cleavable in reflection light, which is reflected on a first beam path in the direction of the light structuring device, and in transmission light, which is passed to a second beam path.

• - Leiten von polarisiertem Licht auf einen Polarisationsstrahlteiler, der das Licht polarisationsabhängig in Reflexionslicht und in Transmissionslicht spaltet, und
• - Leiten des Reflexionslichts vom Polarisationsstrahlteiler auf einem ersten Strahlengang zu einer Lichtstrukturierungsvorrichtung, welche dem Reflexionslicht eine Strukturierung aufprägt.

In a corresponding manner, the following steps are provided in a generic method for providing a structured illumination in a light microscope:

• - Passing polarized light on a polarization beam splitter, which splits the light polarization dependent in reflection light and in transmission light, and
• - Directing the reflection light from the polarization beam splitter on a first beam path to a Lichtstrukturierungsvorrichtung, which imparts a structuring of the reflection light.

In the prior art, one or more gratings are often used as the light structuring device. A grid in an intermediate image plane ensures that incident light is diffracted and structurally receives a grid structure. In the sample plane, this creates a lighting which corresponds to the structure of the grid. The line structure defined by the grid of alternating light and dark areas forms a modulation direction which is perpendicular to the illumination lines. In order to generate the different orientations of the structured illumination, that is to produce different modulation directions of the illumination, the grid can be rotated or differently oriented gratings are illuminated successively. It is also possible to adjust a picture field rotator or to introduce it into the beam path in order to produce different illumination orientations. In these approaches, the relatively large amount of time is disadvantageous. Alternatively, if multiple superposed, differently oriented gratings are used on a substrate, the loss of light output is high. Because unused grid directions / diffraction orders must be hidden so that only one grid direction in the sample space is used for the best possible signal-to-noise ratio. Another known alternative provides to provide an optical beam splitter for generating interfering beams. The arrangement of the interfering rays in a pupil plane is chosen so that it corresponds to the Fourier transform of the desired illumination pattern in the sample plane. However, such designs are relatively complicated and may provide unsatisfactory stability.

As an object of the invention can be considered to provide an optical arrangement for structured illumination for a light microscope and a corresponding method, which allow different modulation directions of the lighting as simple and stable.

This object is achieved by the optical arrangement with the features of claim 1 and by the method with the features of claim 13 solved.

Advantageous variants of the optical arrangement according to the invention and of the method according to the invention are the subject of the dependent claims and are also explained in the following description.

In the optical arrangement of the above-mentioned type, a polarization device is arranged according to the invention in front of the polarization beam splitter such that light is conducted polarized to the polarization beam splitter and impinges there with a polarization direction by which the incident light is neither completely reflected nor completely transmitted. The light structuring device is arranged such that it imparts a structuring which differs from the structuring of the reflection light by the transmission light coming from the polarization beam splitter. The polarization beam splitter is also arranged so that it combines the transmission light and the reflection light, which differ in their structuring and their polarization, on a common beam path. The common beam path can lead in particular in the direction of a sample to be examined.

In an analogous manner, in the method of the abovementioned type, the light-structuring device is arranged according to the invention such that it imparts a structuring on the transmission light coming from the polarization beam splitter, which differs from the structuring of the reflection light. The polarization beam splitter is arranged so that it combines the transmission light and the reflection light, which differ in their structuring and their polarization, on a common beam path.

The invention produces two different modulation directions, in particular simultaneously. In the prior art, however, a pattern, in particular a line pattern, is generated in a sample plane, which only has a periodically fluctuating light intensity in one direction. In the invention, the illumination pattern in two directions is allowed to have a periodically fluctuating intensity because the two overlapping patterns have a different polarization, in which case unwanted interference can be avoided. Instead of simultaneously generating the patterns of different modulation directions, a fast-switching polarization switch can also be provided. By this light of two different polarization directions can successively go through the optics assembly. As a result, speed advantages over conventional methods can also be achieved.

The transmission light and the reflection light each form a lighting pattern. The sample plane may be a line pattern and accordingly one pixel pattern each in a pupil plane. In this case, the transmission light and the reflection light can be mutually perpendicularly linearly polarized. On the one hand, this is important because the polarization direction should be selected appropriately for the direction of modulation. In addition, the perpendicular polarization directions are also important so that no interference occurs between the two overlapping patterns. In the high-frequency modulation range, the signal dynamics are thereby better compared to the case that no different polarization directions would be used.

In an alternative design, a polarization filter follows on the polarization beam splitter, with which the reflection light and the transmission light are brought together. This can be arranged with its polarization axis so that in each case a part of the transmission and reflection light is transmitted. For example, the polarizing filter may be disposed at an angle of 45 ° (or, more generally, 35 ° to 55 °) to each of the polarization directions of the reflection and transmission light. Particularly, in such an orientation, the polarizing filter achieves that a polarization direction of the outgoing light is diagonal to the respective patterns generated by the transmission and reflection light. By way of example, these patterns may each comprise illumination points arranged in a line, the polarization direction being in each case diagonal (45 °) to the line due to the polarization filter. This can be advantageous to achieve an increase in resolution by the structured illumination in the diagonal direction. Otherwise, an increase in resolution would indeed take place in the two modulation directions, but not necessarily in the diagonal direction.

The invention therefore no longer requires that illumination patterns having two different modulation directions be successively irradiated onto a sample; rather, this can be done simultaneously. It is therefore not a fast switch required to switch between lights of different modulation directions.

In order to determine a high-resolution sample image, a plurality of sample images with different phase of the illumination pattern are now taken, and these images are then combined with one another to form a high-resolution image. It is not necessary to generate differently oriented illumination patterns one after the other and in this way to record a plurality of sample images. Although the use of only two modulation directions compared to the usually three successive modulation directions that in the high frequency range of the signal Fourier space is not completely filled in all directions; In contrast, however, there are the considerable speed advantages resulting from the simultaneous generation of two modulation directions.

The polarization beam splitter is a device that transmits or reflects incident light depending on its polarization. In particular, the polarization beam splitter can be formed by a polarization beam splitter cube.

The polarization beam splitter, the light structuring device and beam deflection elements can be arranged so that together with the first and the second beam path a closed loop is formed, which is traversed by the transmission light and the reflection light in the reverse direction. Accordingly, the transmission light, after having been imparted with a structure by the light structuring device, again strikes the polarization splitter, but from the direction in which the reflection light was deflected at the polarization beam splitter. Analogously, the reflection light, after being imparted with a structure by the light structuring device, again strikes the polarization splitter, but from the direction in which the transmission light was transmitted at the polarization beam splitter. In this second impact, the transmission light is transmitted again and the reflection light is reflected again, so that these two are output on a common beam path, in particular in the direction of a sample. So that transmission light is again transmitted to the polarization beam splitter and reflection light is reflected again, the polarization of the light when passing through the closed loop should either not change or change only temporarily, so that the second impingement on the polarization beam splitter, the polarization is the same as when first leaving the polarization beam splitter.

The light structuring device is designed such that a spatial structure is impressed on incident light. For example, the light structuring device may comprise a transmissive or reflective grating as a structured element. In the case of a reflective grating, provision may be made for light to be radiated obliquely thereon so that the input and output directions to the reflective grating are different; This makes it easier to realize a closed loop as a beam path. Alternatively, the light structuring device may also be formed with a liquid crystal matrix, which will be described later.

If, for example, a grating is used as the structured element and both the transmission light and the reflection light strike the grating, this alone does not achieve a different spatial structuring of the transmission and reflection light. This different structuring can be achieved with the help of a Bildrotators. For example, if the transmission light strikes first on the grating and then on the image rotator, the transmission light in a sample plane generates a grating structure illumination rotated on the basis of the image rotator. The reflected light hits the image rotator first and then the grating. The impressed lattice structure of the reflection light is not influenced by the image rotator in this case, so that in a sample plane the reflection light produces a non-rotated lattice structure illumination. Of course, in reverse, the reflection light can first hit the grating and then the image rotator, while the transmission light strikes the image rotator first and then the grating.

The image rotator may also be referred to as a field rotator or rotator. Preferably, a design of an image rotator is selected, which does not rotate the Lichtpolarisation. Alternatively, polarization-influencing means, which compensate for an undesired polarization change of the image rotator, can also be provided on the image rotator.

The structured element should be in focus for both the reflection light and the transmission light. However, due to the image rotator, the optical path length of the beam path is changed from the polarization beam splitter via the image rotator to the structured element. On the other beam path from the polarization beam splitter to the structured element, therefore, the optical path length should be influenced in the same way. This can be done with a transparent retardation element, which can be for example a glass block. Alternatively, the geometrical path lengths for the two beam paths from the polarization beam splitter to the structured element can be selected just as different that the optical path lengths are the same.

The light structuring device may comprise a grating group, which is incident on transmission light and reflection light from different directions. In particular, transmission light may strike a front side of the grating group and reflect light onto a back side of the grating group, or vice versa. A grating group may comprise one or more gratings, wherein a single grating is sufficient to provide structured illumination with two modulation directions in accordance with the invention.

It may be desirable for the two overlapping light patterns of different modulation directions to have the same light intensity as possible. For this purpose, half of the light which strikes the polarization beam splitter should be reflected and half transmitted. This can be achieved if the polarization device is arranged so that a light polarization of the light emitted by it is such that the light at the polarization beam splitter is equally divided between the two beam paths. In principle any optical assembly which outputs (linear) polarized light can be used as the polarization device. This can be a polarizer that only transmits light of a specific polarization direction from incident light. Alternatively, an AOTF (acousto-optical tunable filter) can also be used as polarization device or part thereof. An AOTF comprises a medium, in particular a crystal, in which an acoustic wave is generated, which influences its refractive power. As a result, light of an adjustable wavelength is deflected in the direction of a first diffraction order. The polarization of the first diffraction order can be rotated by 90 ° with respect to the zeroth diffraction order. The polarized light component can be forwarded to the polarization beam splitter. In principle, one or more lasers can simultaneously serve as light source and polarization device in the sense of the invention. Additional optical components are not necessarily required in this case for a polarizing device.

The light structuring device may also include at least one liquid crystal matrix having a plurality of liquid crystal elements independently switchable. The liquid crystal elements may be arranged in a two-dimensional pattern directly next to each other. Such a liquid crystal matrix is also referred to as LCoS or LCoS-SLM (LCoS: Liquid Crystal on Silicon; SLM: Spatial Light Modulator). An adjustable voltage can be applied to each of the liquid crystal elements. The molecules in the liquid crystal element twist depending on the voltage, so that the optical path changes depending on the polarization and thus also changes the optical phase. In this way, a phase of incident light within an interval can in principle be arbitrarily shifted, for example between 0 and 4π. In this case, the polarization direction of the incident light relative to the orientation of the liquid crystal elements determines whether a variable phase change can be set or not. If the polarization of the light is parallel to a direction which is referred to below as the axis of action of the liquid crystal matrix, then the light phase can be variably adjusted, while light with a polarization perpendicular thereto, regardless of a switching state of the liquid crystal matrix, passes through the latter, is reflected at the rear and runs back again, without a phase change would be variably adjustable. In various variants of the invention, such a liquid crystal matrix having a reflective rear side is used, wherein these embodiments can also be modified in principle to form a transmissive liquid crystal matrix which is traversed only once by the light.

By means of a liquid crystal matrix, a phase of an incident light beam can thus be variably adjusted via the beam cross section, given suitable polarization of the light. From this phase grating an amplitude grating / an amplitude variation should result in the sample plane, which represents a structured illumination of bright and dark areas. This is achieved by masking out light fractions: The phase grating corresponds, for example, in a pupil plane several illumination areas, of which the outer are now but not passed through the optical components of the light microscope to the sample. Only one central and two lateral illumination areas / illumination points, which is a 0., -1. and +1. Diffraction order correspond are forwarded to the sample. For this purpose, the liquid crystal elements can be adjusted so that the liquid crystal matrix forms a lattice structure with a lattice constant, which is selected so that only light which is in a pupil plane of a 0..1 -1. and +1. Diffraction order corresponds, reaches the sample. On the other hand, external light components are hidden and do not reach a sample area.

In principle, it is possible to replace the grating by a single liquid crystal matrix in the embodiment described above with a grating and an image rotator. Light is directed obliquely onto the liquid crystal matrix. The transmission or the reflection light can also hit the image rotator after the liquid crystal matrix, so that the structuring differs from the transmission and reflection light.

• Hierzu kann ein Polarisationsdreher zum Drehen der Polarisationsrichtung von auftreffendem Licht um 90° vorhanden und so angeordnet ist, dass er zweimal durchlaufen wird, nämlich direkt vor und direkt nach Auftreffen auf einen der beiden Flüssigkristallbereiche. Durch das zweimalige Auftreffen ist die Polarisationsrichtung schlussendlich gleich wie zuvor, allerdings ist die Polarisationsrichtung beim Auftreffen auf diesen Flüssigkristallbereich um 90° gedreht. So kann bei dieser Ausführung das Transmissionslicht zunächst auf den Polarisationsdreher, dann auf den zweiten Flüssigkristallbereich, dann auf den Polarisationsdreher und dann auf den ersten Flüssigkristallbereich treffen, während das Reflexionslicht in umgekehrter Reihenfolge auf diese Komponenten trifft. Selbstverständlich kann auch hier die Anordnung so abgewandelt werden, dass das Transmissionslicht in umgekehrter Reihenfolge die oben genannten Komponenten durchläuft, während das Reflexionslicht in der genannten Reihenfolge die Komponenten durchläuft.

It may be preferable to use two different liquid crystal regions, wherein one of the two liquid crystal regions influences the transmission light and the other of the two liquid crystal regions influences the reflection light. The two liquid crystal regions may belong to different liquid crystal matrices. In this case, the two liquid crystal arrays may have axes of action rotated by 90 ° with respect to each other so that the different polarization directions of the transmitted light and the reflected light cause only one of the two liquid crystal arrays to variably change the phase of the incident light. However, liquid crystal matrices are very expensive, so that it may be preferable if the two liquid crystal regions are different Are areas of the same liquid crystal matrix. Reflection light is guided on the beam path forming a closed loop from the polarization beam splitter, first to the first liquid crystal region and then to the second liquid crystal region. In this case, the reflection light should have a polarization direction by which the phase of the reflection light is variably influenced only by one of the two liquid crystal regions. On the other hand, the transmission light is first conducted to the second liquid crystal region and then to the first liquid crystal region, having a polarization direction by which the phase of the transmission light is in turn variably affected only by the other of the two liquid crystal regions. The fact that the transmission light and the reflection light are each variably influenced by only one of the two liquid crystal regions can be achieved by suitably rotating the polarization direction of the transmission light and the reflection light, thus requiring no image rotator:

• For this purpose, a polarization rotator for rotating the polarization direction of incident light by 90 ° is present and arranged so that it is traversed twice, namely directly before and directly after hitting one of the two liquid crystal regions. Due to the two-time impingement, the polarization direction is ultimately the same as before, however, the polarization direction is rotated by 90 ° when hitting this liquid crystal region. Thus, in this embodiment, the transmission light may first hit the polarization rotator, then the second liquid crystal region, then the polarization rotator, and then the first liquid crystal region, while the reflected light may encounter these components in reverse order. Of course, here too, the arrangement can be modified so that the transmission light in reverse order passes through the above components, while the reflection light in the order listed passes through the components.

The polarization rotator can be formed by a single λ / 2 plate or comprise two λ / 2 plates, of which one λ / 2 plate is passed before impinging on the second liquid crystal region and the other λ / 2 plate after hitting is passed through the second liquid crystal region. The λ / 2 plates are arranged so that their crystal axes are parallel.

It may be preferred if the transmission light and the reflection light each remain unaffected on the first impact with one of the two liquid crystal regions and are not influenced until the second impact. The liquid crystal regions, the polarization directions of the transmission and reflection light and optionally the optical axis of the polarization rotator can be aligned accordingly. This is conducive to better beam quality.

With the liquid crystal matrix, a lighting pattern can be generated which has two different modulation directions in the sample plane at the same time. As a result, it is not necessary to record different sample images with different modulation directions in succession. Nevertheless, several sample images should be taken in succession with different phase. This can be done by setting different phase modulations with the light structuring device. As an advantage, no components need to be displaced or tilted to effect a phase change, which can provide stability and speed advantages.

If, instead of the liquid-crystal matrix, a grid is used as the structured element, then a phase shift of the illumination light can take place by means of an especially piezotriven movement of the grid / of the structured element. Alternatively, one or more wobble plates can be used, which are adjusted for different images, for example by galvo scanners. The at least one wobble plate can in particular be arranged in an intermediate image plane.

In a modification of the embodiments described above, an amplitude modulation is effected by the two liquid crystal regions. A liquid crystal region alone may cause a phase shift and thereby a polarization rotation or change. Depending on the change in polarity, a ratio between reflection and transmission at the polarization beam splitter, which brings together the transmission and reflection light, can be variably adjusted. The further used light component is thereby modulated in its amplitude / intensity. In this embodiment, no closed loop is formed as a beam path from the polarization beam splitter. Rather, the reflection and transmission light are directed to different liquid crystal regions, preferably the same liquid crystal matrix, and then redirected back to the polarization beam splitter on the same path. In this case, the reflection and transmission light can each be directed perpendicular to the liquid crystal regions. So that they are combined by the polarization beam splitter on a common beam path, the structured transmission light on the polarization beam splitter must be reflected and the structured reflection light must be transmitted. This can be achieved by a through the liquid crystal regions Polarization change is effected. In this case, the transmission and reflection light between the polarization beam splitter and the liquid crystal region each pass through a polarization rotator. This can be run twice, namely both on the way to the liquid crystal region as well as on the way back from this. Due to the liquid crystal regions and the polarization rotator, the polarization direction can be rotated by 90 ° on the way back to the polarization beam splitter. The same polarization rotator or different polarization rotators can be used for the transmission and reflection light. The polarization rotator can be a λ / 2 plate whose optical axis can be at an angle of 22.5 ° to the transmission or reflection light and correspondingly at an angle of 67.5 ° to the other of the transmission or reflection light. Alignment of the liquid crystal array is at an angle of 45 ° to the original polarization direction of both the transmitted and reflected light, and at an angle of 22.5 ° to the optical axis of the λ / 2 plate. This allows the liquid crystal matrix to rotate the polarization direction of the incident light, in particular by 90 °, or to change to circular / elliptical polarization. On renewed passage through the λ / 2-plate so the polarization direction of the transmission light and the reflection light against the initial polarization is rotated by 90 ° or is circularly / elliptically polarized. As a result, it can be adjusted to what proportions the reflection and transmission light are respectively reflected and transmitted at the merging polarization beam splitter.

The liquid crystal regions may be arranged in a pupil plane or an intermediate image plane. In an arrangement in a pupil plane, a phase grating structure impressed here leads to the light in the sample plane having an amplitude grating structure. In order to provide a desired amplitude lattice structure, that is to say a desired lattice intensity distribution in the sample plane, an appropriate phase pattern for the pupil plane can be calculated via an IFTA (iterative Fourier transformation algorithm). A control unit may be configured to determine a phase grating structure for a given desired amplitude grating structure via an IFTA, to which the control unit then adjusts the liquid crystal matrix.

The invention also relates to a light microscope with an optical arrangement, which can be designed as described here. The light microscope comprises a light source connection. At this a light source can be coupled, for example, one or more lasers. The light source connection is designed so that when connecting a light source whose light passes through the beam path described here. In addition, the light microscope may include a detector terminal to which a light detector may be connected. This can be designed as a spatially resolving camera. A polarization-dependent beam splitting is not required in the detection beam path. If, for example, a sample is excited to fluorescence by the structured illumination, then the fluorescent light to be detected is essentially not polarized.

The properties of the invention described as additional optical arrangement features also result in variants of the method according to the invention when used as intended. Conversely, the described components of the optical arrangement can also be set up to carry out the method variants.

• 1 eine schematische Darstellung eines Ausführungsbeispiels eines erfindungsgemäßen Lichtmikroskops mit einem Gitter als strukturiertem Element;
• 2 eine schematische Darstellung eines Ausführungsbeispiels eines erfindungsgemäßen Lichtmikroskops mit einer Flüssigkristallmatrix als strukturiertem Element; und
• 3 eine schematische Darstellung eines Ausführungsbeispiels einer erfindungsgemäßen Optikanordnung mit einer Flüssigkristallmatrix als strukturiertem Element.

Further advantages and features of the invention will be described below with reference to the accompanying schematic figures. Herein show:

• 1 a schematic representation of an embodiment of a light microscope according to the invention with a grid as a structured element;
• 2 a schematic representation of an embodiment of a light microscope according to the invention with a liquid crystal matrix as a structured element; and
• 3 a schematic representation of an embodiment of an optical arrangement according to the invention with a liquid crystal matrix as a structured element.

Identical and identically acting components are generally identified by the same reference numerals in the figures.

1 shows an embodiment of an optical arrangement according to the invention 100 which part of a light microscope according to the invention 110 is.

A light source not shown here 1 sends illumination light 2 , The light source 1 For example, it may comprise a plurality of lasers whose beam paths are brought together by a mirror staircase onto a common beam path.

The optics arrangement 100 includes a light structuring device 30 which imposes a spatial structure on the illumination light. The structured light in this way 52 is about optics components 80 , which in particular a lens 81 to a sample area 83 directed. There generates the structured light 52 an amplitude modulated illumination pattern with alternating bright and dark areas.

Light coming from a sample in the sample area 83 is thrown back as a sample light 82 denotes and can be, for example, luminescent light, ie fluorescence and / or phosphorescent light. It can have the same lens 81 and then passed through a beam splitter 90 from the beam path of the illumination light 52 be separated before passing through a detector 95 is detected.

In the prior art, a line grid illumination is often generated as a lighting pattern in the sample plane. This has exactly one modulation direction, in which a light intensity is modulated. If now several sample images are taken which differ in the light phase, an image can be calculated from these sample images, which has an increased resolution in the direction of modulation. So that not only in this direction the resolution is increased, usually also a plurality of sample images are recorded with a different modulation direction of the illumination pattern, which differ from each other in phase. In the prior art, therefore, a relatively large number of sample images are taken in succession.

Under certain circumstances, the number of required sample images can be reduced by the invention. For this purpose, a lighting pattern is generated which has not only one but two modulation directions in the sample plane. Now several, for example three or more, sample images of different phase can be recorded and calculated into a high-resolution image. An illumination pattern with only one modulation direction in the sample plane corresponds to a dot pattern in a pupil plane, the illumination points being arranged in a line / straight line. According to the invention, a structured illumination is generated which is in a pupil plane 60 a dot pattern 61 generated, whose lighting points 62 - 66 along two (in particular mutually perpendicular) lines are arranged. It is important that the illumination points of the two different lines have different polarization. This makes it possible that the illumination points of each line each generate a modulation direction in the sample plane, without interfering with this separation of the modulation directions. More generally, the two patterns of different polarization can be rotated 90 ° to each other, whereby the illumination points of a pattern need not necessarily lie on a straight line.

• Beleuchtungslicht 2 wird durch eine Polarisationsvorrichtung 5 linear polarisiert. Im dargestellten Beispiel ist oder umfasst die Polarisationsvorrichtung 5 einen AOTF. Mit diesem kann gleichzeitig eine Beleuchtungswellenlänge ausgewählt werden.

The optics arrangement 100 with which such a lighting pattern 61 is explained in more detail below:

• illumination light 2 is through a polarizing device 5 linearly polarized. In the example shown, the polarization device is or comprises 5 an AOTF. With this an illumination wavelength can be selected at the same time.

The linearly polarized light now becomes a polarization beam splitter via a collimator 10 directed. Here are the polarization device 5 and the polarization beam splitter 10 arranged so that the linearly polarized light in reflection light 12A and transmission light 12B is split. reflection light 12A is on a first beam path 11A reflects and simultaneously transmits light 12B on a second beam path 12B transmitted.

The transmission light 12B is over Strahlumlenkelemente 17 , For example, one or more mirrors or prisms, the Lichtstrukturierungsvorrichtung 30 directed. This includes a structured element 39 , which in this example is a grid 39A is and can be arranged in an intermediate image plane. In the example shown, a transmissive grating is used, but it is also possible to use an obliquely illuminated reflective grating. Through the structured element 39 becomes the transmission light 12B applied a spatial structure. The structured transmission light 12B will now have more Strahlumlenkelemente 16 to a picture rotator 50 and on to the polarization beam splitter 10 guided. At the image rotator 50 it will be through the structured element 39 generated illumination pattern rotated, preferably by 90 °. A Lichtpolarisation is thereby by the Bildrotator 50 not co-rotated or alternatively compensated by an additional component, before the structured transmission light 12B on the polarization beam splitter 10 meets. Since the polarization direction of the transmission light 12B is unchanged, it is again at the polarization beam splitter 10 transmitted. The transmission light 12B hits from one direction on the polarization beam splitter 10 into which the reflection light 12A from the polarization beam splitter 10 emanates. As a result, the structured transmission light 12B not in the direction of the light source 1 but in the direction of the sample area 83 ,

The beam path thus forms from the polarization beam splitter 10 a closed loop passing through the structuring device 30 runs. Hereby go through the reflection light 12A and the transmission light 12B the closed loop in the opposite direction. The reflection light 12A therefore, first goes through the image rotator 50 , As the reflection light 12A no structure has been imprinted, has the image rotation of the image rotator 50 no relevant effect on the reflection light 12A , From the image rotator 50 reaches the reflection light 12A , For example via Strahlumlenkelemente 16 , on the structured element 39 and further on, for example, beam deflection elements 17 to the polarization beam splitter 10 , This is the structured reflection light 12A reflects and thus reaches a common beam path 55 with the structured transmission light 12B ,

On the common beam path 55 are the structured transmission light 12B and the structured reflection light 12A perpendicular to each other linearly polarized and have an intensity pattern which is rotated by 90 ° to each other. For a pupil level 60 is the lighting pattern 61 shown. The structured reflection light 12A includes three lighting points 62 . 63 and 66 where the lighting points 62 and 63 one -1 , and +1 , Diffraction order of the structured element 39 correspond and the intermediate illumination point 66 corresponds to a 0. diffraction order. The lighting points 62 . 63 and 66 lie on a straight line and have the same direction of polarization. The structured transmission light 12B also includes three lighting points 64 - 66 , in turn, the lighting points 64 and 65 one -1 , and +1 , Diffraction order of the structured element 39 correspond and the intermediate illumination point 66 corresponds to a 0. diffraction order. The three lighting points 64 - 66 are also on a straight line, which, however, is perpendicular to the line on which the lighting points 62 . 63 . 66 lie.

The transmission light 12B and the reflection light 12A each have a central illumination point 66 , especially exactly on the optical axis. These two lighting points 66 , which correspond to the 0th diffraction order, can be congruent, but differ in their polarization. The 0 , Diffraction order is important for axial modulation of the illumination light.

The dot pattern from the illumination points 62 . 63 . 66 generates in the sample plane a lighting pattern whose amplitude varies periodically in a first direction of modulation. The lighting points 64 . 65 . 66 generate in the sample plane a lighting pattern whose amplitude varies periodically in a second direction of modulation, which is perpendicular to the first direction of modulation.

The optical components 80 and a lattice constant of the structured element 39 can be chosen to each other so that only the lighting points shown 62 - 66 to the sample area 83 while passing through the structured element 39 generated higher order diffraction orders are hidden. This may be desirable for the contrast of the two illumination patterns of different modulation directions.

Through the image rotator 50 is under construction 1 ensures that the two illumination patterns of the transmission and reflection light have different orientations. However, it shifts through the image rotator 50 a focal position of the structuring element 39 continuous reflection light 12A , So that the structured element 39 both for the reflection light 12A as well as the transmission light 12B is located in a focal plane, can in the beam path 11B that the transmission light 12B before hitting the structured element 39 goes through, a delay element 49 be arranged. The delay element 49 is designed to cause the same optical path length change as the image rotator 50 ,

As the first beam path 11A to which the reflection light 12A from the polarization beam splitter 10 is passed, can be understood a beam path, the reflection light 12A to the structured element 39 passes. As a second beam path 12A on which the transmission light 12B from the polarization beam splitter 10 is passed, a beam path can be understood in a corresponding manner, the transmission light 12B to the structured element 39 passes. The transmission light 12B runs on the first beam path 11A to the polarization beam splitter 10 , and the reflection light 12A runs on the second beam path 11B to the polarization beam splitter 10 ,

Optional may be on the beam deflecting elements 16 or 17 can be omitted, or these can be located elsewhere in the beam path forming the closed loop.

Another embodiment of the invention is in 2 shown. The components provided with the same reference numerals as in FIG 1 can be designed as described there. From 1 This embodiment differs in the design of the structuring device 30 , This does not include a grid, but a liquid crystal matrix 35 as a structured element. Such a liquid crystal matrix 35 is also referred to as LCoS (Liquid Crystal on Silicon). Incident light passes through the liquid crystal matrix 35 , is reflected at the back (that is, in particular on the silicon chip) and again passes through the liquid crystal matrix 35 before it leaves. An amplitude modulation of the incident light is not reached here alone, but a phase modulation. The liquid crystal matrix 35 includes several liquid crystal elements that are birefringent and can be adjusted independently. Depending on the setting, a liquid crystal element can variably change the phase of incident light, but only if the polarization direction of the incident light is suitable for the liquid crystal matrix. In the case of a perpendicular polarization direction, however, the light is passed on without switching states of the liquid crystal elements having an influence on a phase change of the light.

In the example of 2 meet the reflection light 12A and the transmission light 12B on the same liquid crystal matrix 35 , Because the reflection light 12A and the transmission light 12B mutually perpendicularly linearly polarized, would be variable phase-modulated without further action only either the reflection or the transmission light. So that both the reflection and the transmission light can be variably phase-modulated, a polarization rotator is used 28 used. This may be a λ / 2 plate oriented to rotate the polarization of incident transmission or reflection light by 90 °.

In the example shown, the reflection light hits 12A first on a first area 35A the liquid crystal matrix 35 , Then it is a deflection element 18 For example, a mirror or a prism, deflected and is a second time on the liquid crystal matrix 35 directed. But before the second time on the liquid crystal matrix 35 meets, it hits the polarization rotator 28 , which rotates the polarization direction by 90 °. The reflection light 12A This has a different polarization at the second impact than at the first impact. Thus, the reflection light becomes 12A only at the first or the second impact variable phase modulated, while it undergoes no phase modulation at the other impact.

After the second impact on the liquid crystal matrix 35 goes through the reflection light 12A again the polarization rotator 28 , whereby the polarization direction is turned back again.

At the first impact, the reflection light strikes a first liquid crystal region 35A the liquid crystal matrix 35 and the second time to a second liquid crystal region 35B which in particular is different or non-overlapping to the first liquid crystal region 35A can be.

The transmission light 12B goes through the same beam path as the reflection light 12A but in the opposite direction. Thus, the transmission light hits 12B first on the polarization rotator 28 before it reaches the liquid crystal area 35B for the first time on the liquid crystal matrix 35 meets. Then it goes through the polarization rotator again 28 and then strikes the liquid crystal region 35A ,

The reflection light 12A thus becomes only the first or second liquid crystal region 35A or 35B phase modulated while the transmission light 12B is phase modulated by the other of the two liquid crystal regions. This allows the reflection light 12A and the transmission light 12B different phase modulations are impressed.

The liquid crystal elements of the first and second liquid crystal regions 35A . 35B can be set to produce phase gratings. A lattice constant of this phase grating can now be chosen so that higher diffraction orders are not affected by the optical components 80 to the sample area 83 be routed, but previously hidden. Alone a -1, 0 and +1. Diffraction order will be up to the sample area 83 directed. As a result, in the sample plane from the original phase grating an amplitude grating, so an intensity modulation.

In a pupil plane 60 superimpose the structured reflection light 12A and the structured transmission light 12B to a dot pattern 61 , This can be the same as with 1 be.

Through the liquid crystal matrix 35 can advantageously be generated a variably adjustable phase modulation. If several sample images of different phase are to be recorded successively, this can be done by switching the liquid crystal matrix accordingly 35 respectively. There are no mechanical shifts or rotations of components necessary and it also does not have different beam paths to be selected successively. As a result, a speed advantage can be achieved and the stability to shocks can be greater.

In the execution of 2 are in the beam path of the closed loop an image rotator 50 and a delay element 49 not mandatory.

In a modification of the example of 2 can the liquid crystal areas 35A and 35B be formed by the same area or overlapping areas. It can be provided that the light at the first and second impingement at different angles on the liquid crystal matrix 35 meets. In this case, the liquid crystal matrix generates 35 the same phase modulation for the transmission light and the reflection light. Therefore, in this modification, the image rotator is important to output differently oriented modulation patterns. In such a design, the dimensions of the polarization rotator 28 do not bother; For this purpose, in particular further Strahlumlenkelemente between the polarization rotator 28 and the liquid crystal matrix 35 be arranged. As a possible advantage of this embodiment, a smaller liquid crystal matrix is sufficient.

In a further modification, the illustrated liquid crystal matrix 35 be replaced by two liquid crystal matrices. If these liquid-crystal matrices are arranged perpendicular to one another in their effective direction, the polarization rotator can be dispensed with. However, since liquid crystal arrays are very expensive, this design is associated with higher costs.

In another modification of the embodiment of 2 can the polarization rotator 28 and the deflecting element (s) 18 may also be formed by a common assembly, for example by a video field rotator, which also rotates the polarization or includes a corresponding component thereto.

3 shows a schematic plan view of an optical arrangement according to the invention 100 which is essentially the same as the optics assembly 2 can be. In 3 are with arrows the propagation directions of the illumination light 2 , the reflection light 12A and the transmission light 12B shown. In addition, here is the polarization direction of the transmission light 12B indicated on the different beam path sections. As shown, changes through the polarization rotator 28 the polarization direction twice. The polarization direction of the reflection light 12A is perpendicular to that of the transmission light on each optical path section 12B and is not in for clarity 3 entered. On the common beam path 55 are both polarization directions of the reflection light 12A and the transmission light 12B entered.

LIST OF REFERENCE NUMBERS

1

light source

2

illumination light

5

Polarization device, AOTF

10

Polarization beam splitter

11A

first ray path

11 B

second beam path

12A

reflection light

12B

transmission light

16, 17, 18

deflecting

28

The rotator

30

structuring device

35

liquid crystal matrix

35A

first area of the liquid crystal matrix

35B

second region of the liquid crystal matrix

39

structured element

39A

grid

49

delay element

50

image rotator

52

structured illumination light

55

common beam path

60

pupil plane

61

(Lighting) dot pattern

62-66

lighting points

80

optical components

82

sample light

83

sample area

90

beamsplitter

95

detector

100

optics assembly

110

light microscope

Claims (16)

A structured illumination optical arrangement for a light microscope, comprising - a light structuring device (30) imparting structuring to incident light, - a polarization beam splitter (10) arranged so that incident light (2) is polarization-dependent in reflection light (12A) a first beam path (11A) in the direction of the light structuring device (30) is reflected, and in transmission light (12B) on a second beam path (11B) is cleavable, characterized in that before the polarization beam splitter (10) has a polarizing device (5) is arranged in that light (2) is conducted in a polarized manner to the polarization beam splitter (10) and impinges there with a polarization direction by which the incident light (2) is neither completely reflected nor completely transmitted, - the light structuring device (30) is designed so that come from the polarization beam splitter (10) the transmission light (12B) is imprinted with a structuring which differs from the structuring of the reflection light (12A), - the polarization beam splitter (10) is arranged so that it transmits the transmission light (12B) and the reflection light (12A) distinguished structuring and their polarization, merges on a common beam path (55). Optics arrangement after Claim 1 , characterized in that the structuring device (30) imparts structuring to the reflection light (12A) and the transmission light (12B) such that in a pupil plane (60) in the common beam path (55): - the reflection light (12A) has illumination points (62, 63, 66) lying on a straight line, and - the transmission light (12B) forms illumination points (64, 65, 66) lying on a straight line perpendicular to the straight line of the illumination points (62, 63, 66) of the reflection light (12A), wherein a polarization direction of the reflection light (12A) is perpendicular to a polarization direction of the transmission light (12B). Optics arrangement after Claim 1 or 2 , characterized in that the polarization beam splitter (10), the light structuring device (30) and Strahlumlenkelemente (16, 17) are arranged so that with the first and the second beam path (11A, 11B) together a closed loop is formed, which of the transmission light (12B) and reflected light (12A) is reversed. Optics arrangement after Claim 2 or 3 characterized in that the light structuring device (30) comprises a patterned element (39) and an image rotator (50) disposed in the closed loop such that one of the transmission light (12B) and reflection light (12A) is first applied to the image rotator (50) and then hits the patterned element (39), while the other of the transmission light (12B) and reflection light (12A) strikes first the patterned element (39) and then the image rotator (50), in particular the first Beam path (11A) to the structured element (39) and the second beam path (11B) to the structured element (39) are designed so that despite image rotator (50), the optical path lengths from the polarization beam splitter (10) to the structured element (39) are the same in particular in that a transparent delay element (49) is arranged in the first or second beam path (11A) whose optical path length is equal to an optical path length of the optical path Image rotator (50), and the image rotator (50) is arranged in accordance with other of the two beam paths (11B). Optics arrangement according to one of Claims 1 to 4 , characterized in that the light structuring device (30) comprises a grating group (39A) to which transmission light (12B) and reflection light (12A) strike from different directions. Optics arrangement according to one of Claims 1 to 4 , characterized in that the light structuring device (30) comprises at least one liquid crystal matrix (35) having a plurality of independently switchable liquid crystal elements with which a phase of incident transmission light (12B) and reflection light (12A) is adjustably changeable. Optics arrangement after Claim 6 characterized in that said at least one liquid crystal array (50) comprises first and second liquid crystal regions (35A, 35B), said reflection light (12A) being incident on said optical path forming a closed loop from said polarization beam splitter (10) first liquid crystal region (35A) and then onto the second liquid crystal region (35B), thereby having a polarization direction by which the phase of the reflection light (12A) is variably influenced only by one of the two liquid crystal regions (35B), the transmission light (12B) first to the second liquid crystal region (35B) and then to the first liquid crystal region (35A), thereby having a polarization direction by which the phase of the transmission light (12B) is variably affected only by the other of the two liquid crystal regions (35A). Optics arrangement after Claim 7 characterized in that the first and second liquid crystal regions (35A, 35B) are portions of the same liquid crystal matrix (35), that a polarization rotator (28) for rotating the direction of polarization of incident light is disposed at 90 ° and arranged so that one of the transmissions - and the reflection light (12B): first on the polarization rotator (28), then on the second liquid crystal region (35B), then again on the polarization rotator (28) and then on the first liquid crystal region (35A) meets, while the other from the transmission - And the reflection light (12A) in reverse order on these components (28, 35A, 35B) meets. Optics arrangement after Claim 7 or 8th characterized in that the liquid crystal regions (35A, 35B) and the polarization directions of the transmission light (12B) and the reflection light (12A) are aligned such that each of the transmission light (12B) and the reflection light (12A) is incident upon first impingement the two liquid crystal regions (35A, 35B) remain unaffected and are influenced on the second impact on the liquid crystal regions (35A, 35B). Optics arrangement after Claim 6 , characterized in that the reflection light (12A) from the polarization beam splitter (10) via a Polarization rotator, in particular a λ / 2-plate, on a first liquid crystal region (35A) of the liquid crystal matrix (35) is passed and from there in the same way again via the polarization rotator to the polarization beam splitter (10) is passed, wherein the polarization rotator is arranged so that it rotates the polarization of the reflection light (12A) by 90 °, so that the reflection light (12A) is now transmitted to the polarization beam splitter (10) that the transmission light (12B) from the polarization beam splitter (10) via the same or an additional polarization rotator on a second liquid crystal region (35B) of the liquid crystal matrix (35) is passed from there in the same way back to the polarization beam splitter (10) via said polarization rotator, the polarization rotator being arranged to reduce the polarization of the transmission light (90B) by 90 ° rotates, so that the transmission light (12B) now a m polarization beam splitter (10) is reflected on the common beam path (55) with the reflection light (12A). Optics arrangement according to one of Claims 1 to 10 , characterized in that on the common beam path (55), a polarizing filter is arranged, which is arranged diagonally to the polarization directions of the reflection light and the transmission light. Light microscope with an optical arrangement according to one of Claims 1 to 11 , Method for providing structured illumination in a light microscope, comprising: - directing polarized light (2) onto a polarization beam splitter (10) which splits the light (2) polarization-dependent into reflection light (12A) and transmission light (12B); Reflection light (12A) from the polarization beam splitter (10) on a first beam path (11A) to a light structuring device (30) which imparts a structuring to the reflection light (12A), characterized in that - the light structuring device (30) is arranged so that it is from Polarization beam splitter (10) is imparted a structuring which differs from the structuring of the reflection light (12A), that the polarization beam splitter (10) is arranged such that it transmits the transmission light (12B) and the reflection light (12A). , which differ in their structuring and their polarization, to one common beam path (55) merges. Method according to Claim 13 , characterized in that the polarization device (5) is arranged so that a light polarization of the light emitted by it is such that the light at the polarization beam splitter is equally divided between the two beam paths (11A, 11B). Method according to Claim 13 or 14 , characterized in that the light structuring device (30) comprises at least one liquid crystal matrix (35), with which a phase modulation of incident light is set such that successively a plurality of sample images are taken in which different phase modulations are set with the light structuring device (30). Method according to Claim 15 , characterized in that liquid crystal elements of the liquid crystal matrix (35) are adjusted so that the liquid crystal matrix (35) forms a lattice structure with a lattice constant which is selected so that only light which is in a pupil plane (60) of a 0..1 , or +1. Diffraction order corresponds, is forwarded to a sample area (63), while further outward light components are hidden.

Images