Illumination device with a deflection device and microscope

Abstract

The invention relates to an illumination device (1) for illuminating a sample (8) with an illumination beam along an illumination beam path (B), wherein an adjustable optical deflection device (2) is arranged in the illumination beam path (B) in or near an intermediate image plane (ZB). Characteristically, the deflection device (2) is designed as a combination of at least one first and one second optical element (2.1, 2.2) in the form of optical lenses or optical wedges, of which at least one of the optical elements (2.1, 2.2) is adjustable with respect to its rotational position, its axial position and / or its lateral position, relative to the optical axis (oA) of the illumination beam path (B), compared to the other optical element (2.1, 2.2).2) is adjustable, wherein, by the action of the deflection device (2), at least one beam of the illumination radiation is or can be positioned in a projection plane (PB) extending perpendicular to the illumination beam path (B) in a targeted manner laterally to the optical axis (oA), wherein the projection plane (PB) can optionally be used as an entrance pupil (EP) of an objective (7), and a control (11) is provided by means of which the deflection device (2) can be controlled. The invention also relates to a microscope with an illumination device (1).

Description

The invention relates to an illumination device according to the preamble of the independent claim and to a microscope with such an illumination device. The illumination device is particularly intended to be used in combination with a wide-field microscope to achieve oblique illumination and / or structured illumination of a sample. Several technical solutions are known from the prior art for directing illumination radiation onto a sample in a sample chamber at selected angles. Such illumination methods, for example HILO (highly inclined and laminated optical sheet) illumination, can be used when only a selected area of ​​the sample needs to be illuminated. It is possible to use a single objective lens in a dual function as both an illumination and detection objective. A special form of oblique illumination is microscopy using total internal reflection (TIR), which is employed in the imaging method of total internal reflection fluorescence microscopy (TIRF). In this technique, the illumination radiation is directed in a beam at a very shallow angle onto an interface, such as a glass surface (e.g., a sample holder or coverslip), between the objective lens and the sample. The illumination angle is equal to or less than the angle of total internal reflection. The illumination radiation thus undergoes total internal reflection at the interface with the sample. This creates an evanescent field in the direction of the sample, which can excite fluorescent markers in a region near the sample surface to emit fluorescence radiation.This fluorescence radiation can be detected as radiation and used to image the corresponding areas of the sample. Oblique illumination, or TIR illumination (hereinafter also referred to simply as TIRF illumination), can be achieved using a variety of technical solutions. A beam of illumination radiation is directed into a spot within the objective pupil, i.e., the entrance pupil of an illumination objective, at a point laterally offset from the objective's optical axis. To achieve the desired illumination effect within the sample space, the spot must be positioned very precisely. Furthermore, the illumination objective must have a numerical aperture greater than the refractive index of the sample. In microscopes suitable for TIRF, the spot positioning is achieved, for example, by tilting a mirror or a linear phase pattern in a plane optically conjugated to an intermediate image or to a sample plane. Other methods that can be used include spatial light modulators (SLM; US 9,915,815 B2), moving optical fibers (DE 102 299 35 B4, US 2004 / 001253 A1), plane-parallel glass plates near an optically conjugated pupil plane (US 2014 / 0104680 A1), reflections (DE 102 589 45 A1), or optical fibers in combination with a collimator (DE 10 2008 284 490 A1). In contrast, the SIM method is based on oblique illumination of the sample with at least two beams of light that are also oblique to each other. Interference between these beams creates a structured illumination within the sample, which interacts with the corresponding fluorescent dyes present. It has proven practical to use the diffraction orders of a phase grating (hereinafter also referred to simply as grating) to generate a pattern. Typically, only the lowest diffraction orders of the grating are used, in particular the 0th and the + / -1th diffraction orders. For this purpose, a fixed grating (static variant) or a variable grating, for example in the form of a selective laser ray (SLM) (dynamic variant), can be arranged in an intermediate image plane. The diffraction orders of the grating are imaged as points (spots) into the entrance pupil of the objective lens. In a subsequent sample plane, a resulting illumination pattern is created by interference of the superimposed beams. Alternatively, an amplitude grating can be imaged into the sample plane. If incoherent light is to be used, an amplitude grating must be employed.However, due to the light losses occurring at an amplitude grating, a phase grating is preferable. For the two aforementioned illumination methods, TIRF and SIM, it is crucial that the light is optimally focused into the entrance pupil of the lens. A collimator, for example in the form of a movable or variable and / or electronically controlled lens, can be used to achieve correct focus adjustment. As outlined above, many current TIRF illumination designs shift either the light source or its image to adjust the desired position within the entrance pupil of the illumination lens. If the devices incorporating this type of illumination are to offer other illumination options, an additional intermediate image plane must be created, increasing both the technical requirements and the space required. Using a mirror to deflect the illumination radiation has the disadvantage that any contamination on the mirror will be noticeable twice due to the double transmission of light. If, on the other hand, a deflecting prism is used to deflect the light beam, a mirror can be dispensed with, but a variable adjustment of the deflection angles is not possible. When using the SIM principle for illumination via a phase grating in the intermediate image, the position of the diffraction orders in the entrance pupil depends on the wavelength of the illumination radiation. Therefore, the entire pupil cannot be utilized, and thus the maximum resolution increase cannot be achieved when a single grating for multiple wavelengths is used. The object of the invention is to reduce the aforementioned disadvantages in the technical implementation of the TIRF and SIM illumination methods. Furthermore, it aims to provide a method by which both illumination variants can be made available in a single device. The problem is solved with a lighting device according to claim 1 and a microscope according to claim 10. Advantageous embodiments are the subject of the dependent claims. The invention relates to an illumination device for illuminating a sample with an illumination radiation along an illumination beam path, wherein an adjustable optical deflection device is arranged in the illumination beam path in or near an intermediate image (intermediate image plane). The lighting device according to the invention is characterized in that the deflection device is designed as a combination of at least one first and one second optical element in the form of optical lenses or optical wedges. An embodiment with only two optical elements keeps the number of installed and potentially controllable components to a minimum. In the following, reference is made to two optical elements, without thereby excluding variants with more than two optical elements. Of the two optical elements, at least one is adjustable with respect to its rotational position, its axial position and / or its lateral position relative to the other optical element. The axial and lateral positions are considered relative to the optical axis of the illumination beam path and to the other optical element. The rotational position is defined in relation to the two optical elements relative to each other, whereby the illumination radiation is influenced by both optical elements. The deflection device deflects at least one beam of the illumination radiation into a projection plane extending perpendicular to the illumination beam path, positioned laterally to the optical axis. This deflection can be achieved, or the beam can be positioned in this way. This effect can optionally be achieved in conjunction with other optical components of the illumination beam path. The projection plane could, for example, be the entrance pupil of an illumination lens. A control unit, such as a computer, microcontroller, or FPGA, is provided to control the deflection device. The invention thus essentially proposes a lighting device that allows controlled adjustment of the lighting radiation in a defined plane with only a few components, thereby significantly reducing disadvantages of the prior art, such as a complicated structure, high space requirements, an undesirable wavelength dependence of usability and susceptibility to contamination. To generate desired illumination, for example of a sample, starting from the illumination radiation directed into the projection plane according to the invention, a further embodiment of the invention may include an illumination lens whose entrance pupil (also: objective pupil) lies in the projection plane. Depending on the outline of a beam of light formed into a spot at the entrance pupil, the illumination lens directs the light radiation into a sample chamber and onto a sample optionally arranged therein. If a spot is placed off-axis in the projection plane or off-axis with the entrance pupil, the beam of light is directed into the sample chamber at an illumination angle relative to the optical axis and at a specific azimuthal angle.The positioning of a spotlight in the entrance pupil, in conjunction with the lighting lens, therefore determines the angle and direction of illumination. The deflection device can be designed in various ways. In one alternative, it is designed as a combination of optical lenses in the form of optical wedges, configured as a pair of rotating wedges. In a second alternative, the deflection device can be designed as a swivel wedge (swivel wedge, pair of swivel lenses) or as a pair of sliding lenses. Optical wedges are specially shaped prisms. Their deflection angle δ is determined by the wedge angle α and their refractive index n. For small wedge and incidence angles, the following applies: In a pair of rotating wedges, two, usually identical, prisms are arranged sequentially along an optical axis of a beam path and can be rotated in opposite directions around the optical axis. Depending on the relative rotation of the wedges, the length of the glass path that the individual beams of the illumination radiation must travel inside each wedge changes. If the wedges are rotated 180° relative to each other, the glass path is the same length for all beams parallel to the optical axis. Furthermore, changes in the direction of the beams caused by refraction cancel each other out. If the wedges are in a different rotation relative to each other, the glass paths and the refractive effects of the wedges on the individual beams differ. A maximum deflection results from the sum of the deflections of the individual wedges δi ≈ (n - 1)α (see, for example, Fig. 3 and Fig. 4). For arbitrary rotations ϕ of the wedges relative to each other, the following applies: In an Abat wedge, a variable deflection is generated by means of a plano-concave lens. The curved surface of a plano-convex lens, rotatable relative to the plano-concave lens, is inserted into the spherical or cylindrical surface of this lens. The plano-convex lens has the same radius and refractive power (see also Fig. 5). It is important that the distance of the center of rotation from the curved surface of one of the optical elements corresponds to the radius of that element, so that the distance between the inner spherical surfaces of the optical elements (lenses) remains constant for every rotational position. Depending on the distance between the two optical elements, for example, depending on the size of the gap between the curved surfaces, the optical effect of both lenses almost cancels out, or a slight residual lensing effect of the double-lens system remains. A pair of sliding lenses, like the Abat wedge, consists of a plano-concave (diverging lens) and a plano-convex lens (converging lens, see also Fig. 1 and Fig. 2). In this arrangement, however, the lenses are shifted laterally relative to each other. If only one of the two lenses is shifted, the deflection angle δ depends on the lateral shift x0 and the focal length f as follows: Using the optical transfer function of the deflection group, the effect of the two diametrically shifted lenses on the illumination beam can be well analyzed. The deflection device, here consisting of the converging lens and the diverging lens, represented by the term ei δ·x, causes the deflection of one or more beams of light, as shown by the linear phase. The lateral displacement x0 influences the slope of the linear phase and thus the beam deflection. In a pair of sliding lenses, both optical elements can be moved in opposite directions (diametrically) in a lateral direction, i.e., perpendicular to the optical axis. For the purposes of this description, it suffices if one of the optical elements is moved laterally relative to the other. The lateral displacement of the optical elements to the optical axis results, in simplified terms, in the light rays passing through two glass wedges, with the wedge of a converging lens and the wedge of a diverging lens exhibiting opposite curvatures. As a result, the curvatures cancel each other out, but the wedge effects add up, and the light rays are deflected accordingly. The procedure of moving both optical elements of a pair of sliding lenses in opposite directions and by the same amount perpendicular to the optical axis also allows aberrations caused by the optical elements to be better compensated. As already mentioned, the first and second optical elements can be moved by means of rotation or translation. In the case of an embodiment as a pair of sliding lenses, the movement can be opposite and lateral to each other. In a further embodiment of the invention, it is also possible for only one of the optical elements to be moved laterally to the optical axis. The rotations or translations serve to set a desired deflection angle, i.e., to position beams of light laterally to the optical axis. Furthermore, to position the beams, particularly when the illumination radiation comprises several wavelengths, also in the direction of the optical axis, i.e., axially and parallel to the optical axis, the illumination device according to the invention advantageously includes a collimator. This is arranged upstream of the deflection device and is preferably adjustable. The term "positioning" refers in particular to the location of a spot or focus of the beams. The collimator is intended to reduce or completely eliminate wavelength-related (chromatic) axial aberration. To achieve such a collimator function in an alternative embodiment, at least one of the two optical elements, in particular a pair of sliding lenses, can be designed as an adaptive lens. The focal length of an adaptive lens can be adjusted as needed. An adaptive lens can, for example, be a liquid lens, a spatial light modulator, or a liquid crystal display (LCD). In a further embodiment of the lighting device according to the invention, a flat plate, pivotable about at least one axis of rotation perpendicular to the optical axis, can be arranged upstream of the deflection device in the illumination beam path. This plate is transparent and refracting for at least certain wavelength ranges of the illumination radiation. Such a flat plate allows the beam position of the illumination radiation in the illumination beam path to be adjusted and / or corrected using simple means. Depending on the orientation of the flat plate in the illumination beam path, the beams can be aligned so that they, for example, strike the deflection device parallel to and / or centered on the optical axis. The embodiments of the illumination device according to the invention described so far can be used, for example, to adjust the positioning of at least one beam of light in the projection plane or in the entrance pupil of the lens. In particular, TIRF illumination and HILO illumination can be generated, depending on the position at which the at least one beam of light strikes the projection plane. To generate SIM illumination, an optical grating can be placed upstream of the deflection device. This grating splits the illumination radiation into multiple beams of different diffraction orders, which can then be positioned in the projection plane by the deflection device. Advantageously, the zeroth and the first diffraction orders are projected onto the projection plane. If the entrance pupil of the illumination objective is positioned in this plane, an illumination pattern is generated in the sample space or on or within the sample. The lighting device can be used, for example, to generate TIRF and / or HILO illumination, or SIM illumination, if the optical grating can be removed from or inserted into the illumination beam path as needed. This can be done manually, but advantageously by motor. Equivalent to this is the presence of, for example, a spatial light modulator (SLM) that can produce the effects of a grating. The SLM can be switched to neutral mode and therefore does not need to be removed from the illumination beam path when HILO or TIRF illumination is required. It is also possible to leave the grating in its active state within the illumination beam path while oblique illumination is generated. In such a case, oblique illumination with an illumination pattern can be created in the sample room. The illumination device according to the invention can be a component of a microscope, in particular a wide-field microscope. Such a microscope can have an illumination device according to one of the specified embodiments for selectively illuminating a sample with illumination radiation, at least in the first operating mode of oblique illumination and in the second operating mode of structured illumination. The optical grating can optionally be inserted into and removed from the illumination beam path. In the second operating state, the optical grating is inserted into the illumination beam path or is in the active state. The beams are directed, for example, radially symmetrically around the optical axis into the entrance pupil. This is synonymous with a change in the grating's effect between a neutral setting (neutral state) and an effect similar to that of an optical grating (active state). In the first operating state, the optical grating is removed from the illumination beam path or switched to neutral. The beams of light are directed towards the entrance pupil off-axis. The invention is explained in more detail below with reference to exemplary embodiments, figures, and equations. The figures show: Fig. 1 a schematic representation of a pair of sliding lenses in a neutral position; Fig. 2 a schematic representation of the pair of sliding lenses in a working position with optical elements diametrically offset from each other; Fig. 3 a schematic representation of a pair of wedges in a neutral position; Fig. 4 a schematic representation of the pair of wedges in a working position with the optical elements aligned in the same direction; Fig. 5 a schematic representation of a first embodiment of an illumination device according to the invention as a component of a microscope; Fig. 6 a schematic representation of a second embodiment of an illumination device according to the invention as a component of a microscope; Fig.Fig. 7 a schematic representation of a third embodiment of an illumination device according to the invention as a component of a microscope; Fig. 8 a schematic representation of a fourth embodiment of an illumination device according to the invention as a component of a microscope; and Fig. 9 a schematic representation of a projection plane or entrance pupil of an illumination objective with depicted beams of zeroth and first diffraction order. Unless otherwise stated, the reference numerals in the following exemplary embodiments denote the same technical elements in all illustrations. Figures 1, 2, 3 to 4 illustrate the basic operating principle of a deflection device 2. The resulting effects of the optical elements 2.1 and 2.2 on an illumination beam are shown schematically and by way of example. Figures 1 and 2 show a deflection device 2 in the form of a pair of sliding lenses with two optical lenses. The first optical element 2.1 is a plano-convex lens, and the second optical element 2.2 is a plano-concave lens, arranged along a beam path, illustrated with its optical axis oA. The optical effects of both optical elements 2.1 and 2.2 cancel each other out in the operating position shown (Figure 1). An illumination beam (represented by an arrow) passes through the deflection device 2 without deflection. If the optical elements 2.1 and 2.2 are laterally displaced relative to each other in a further operating position, the neutral effect of the deflection device 2 is eliminated, and the illumination radiation, represented by a single beam, is deflected away from the optical axis oA by a deflection angle δ, corresponding to the displacement and the glass paths the beam must traverse and the refractions occurring. In the example shown in Fig. 2, the first optical element 2.1 is displaced upwards by a distance x0, and the second optical element 2.2 is displaced downwards by a distance x0 in the opposite direction. The resulting deflection δ is shown in a simplified manner as the result of the effects of both optical elements 2.1 and 2.2. In an optional embodiment of the deflection device 2, the focal length of the first optical lens 2.1 or the second optical lens 2.2 can be controlled. The first optical lens 2.1 or the second optical lens 2.2 can be designed as an adaptive lens. This allows the deflection device 2 to generate both an adjustable optical wedge and an adjustable defocus. Adaptive lenses can also produce negative focal lengths. It is also possible that both the first optical lens 2.1 and the second optical lens 2.2 are designed as adaptive lenses. In other possible embodiments of the deflection device 2, only one of the optical elements 2.1, 2.2 can be laterally displaced. Furthermore, the distances x0 need not necessarily be equal if the displacement is performed in the opposite direction. In such a case, with reference to equation 4, a quadratic term remains, which corresponds to a defocus. Correction is possible, for example, using an adjustable optical lens. Figures 3 and 4 are to be understood analogously to Figures 1 and 2 with regard to their mode of operation. Figure 3 shows a pair of optical wedges in a top view. The wedges, functioning as the first optical element 2.1 and the second optical element 2.2, are shown in Figure 3 in a working position with a neutral effect. The length of the glass paths to be traversed is the same for each beam of light propagating parallel to the optical axis oA. The effects of any refractions that occur cancel each other out. Figure 4 shows the optical elements 2.1 and 2.2 aligned in the same direction. Beams of light parallel to the optical axis oA must now travel different lengths through the glass depending on their lateral position. Furthermore, the changes in direction caused by refraction do not cancel each other out. As a result, the beams of light are deflected by an angle δ, depending on their lateral position. The degree of deflection by the optical elements 2.1, 2.2 shown by way of example in Figs. 1, 2, 3 to 4 depends, for example, on the material, in particular the refractive power, of the optical elements, as well as their shape. For the sake of simplicity, the optical elements 2.1, 2.2 of the sliding lens pair can be considered wedges for each beam of light. The deflection angle δ also depends on a wedge angle, i.e., an inclination of the side surfaces of the optical elements 2.1, 2.2 in the region where a beam of light passes through them. An application of the previously described principle in a first embodiment of an illumination device 1 according to the invention as a component of a microscope M is described in Fig. 5. An illumination beam, symbolized by short, solid lines, passes sequentially through an illumination beam path B to a first optical lens 4.1 and a second optical lens 4.2. The second optical lens 4.2 is axially adjustable, i.e., along the optical axis oA, and together with the first optical lens 4.1, functions as a collimator. This allows the axial positioning of beams of light, even those of different wavelengths, to be adjusted in a projection plane PB. The illumination radiation is deflected laterally to a desired extent relative to the optical axis oA by means of a controlled and adjustable deflection device 2. The deflection device 2 is located in or near an intermediate image plane ZB and is designed as an Abatsche wedge. The intermediate image plane ZB is optically conjugate to a sample plane 9. The first optical element 2.1 is a plano-concave lens, and the second optical element 2.2 has a plano-convex shape. The concave and convex surfaces of the optical elements 2.1 and 2.2, respectively, face each other. The second optical element 2.2 can be pivoted relative to the first optical element 2.1 about an axis of rotation perpendicular to the optical axis oA. The resulting changes in the glass path across the cross-section of the illumination beam path B lead to a deflection of beams of the illumination radiation.The illumination radiation is directed, and in particular focused, onto the projection plane PB by a tube lens 5, into which an entrance pupil EP (also: objective pupil) of an objective 7 is positioned. Prior to this, the illumination radiation has passed through a dichroic color splitter 6, which is transparent to the illumination radiation and performs the function of a (primary) color splitter in the microscope M. In further embodiments of the invention, the beam paths can be aligned to each other in such a way that the dichroic color splitter 6 is highly reflective for the illumination radiation and transparent for the detection radiation. The illumination radiation, or at least one beam thereof, enters the entrance pupil EP off-axis oA. Through the action of the lens 7, which acts as an illumination objective, the illumination radiation is directed as oblique illumination with an illumination angle ε other than 0°, measured as the angle between a normal to the sample plane 9 and the beam, onto a sample 8 located there. Depending on the positioning of the beam in the projection plane PB or in the entrance pupil EP, and depending on the optical properties of the surface of the sample 8, an oblique illumination, for example HILO or TIRF illumination, is produced.The latter occurs when the illumination angle ε is larger than the total internal reflection angle of an interface, for example, a glass of a sample holder (only indicated) between the objective 7 and the sample 8, and total internal reflection of the illumination radiation occurs at the interface of the glass. If the illumination radiation is directed along the optical axis oA into the entrance pupil EP, the sample 8 is subjected to wide-field illumination (epi-illumination) (see, for example, Fig. 7 and Fig. 8). In this example, detection radiation from sample 8 is also collected by objective 7, which now functions as the detection objective. The collected detection radiation (a broken solid line with a longer division) reaches the color splitter 6, which is reflective for the wavelength(s) of the detection radiation. From the main color divider, the color divider 6, the detection radiation passes to a tube lens 5 in the detection beam path D and is focused by this lens onto a detector 10 (camera 10). Based on the detection radiation captured there, the detector 10 provides measured values, advantageously with spatial resolution, which can be supplied to an evaluation unit. The evaluation unit can be implemented separately or as part of a controller 11, for example as a compartment. The controller 11 is connected in a manner suitable for data transmission to drives (not shown) of the deflection device 2 and optionally to the detector 10 and / or to a light source 3 (see Fig. 6, Fig. 7 to Fig. 8). Optionally, the control unit 11 can be set up to control the deflection device 2, the light source 3 and / or the second optical lens 4.2 depending on the results of the evaluation unit and to adjust them after manual input by a user or in the sense of a feedback control. In a second embodiment of the lighting device according to the invention, the illumination radiation passes through a light-conducting fiber as a light source 3 to optical lenses 4, 4.1 and 4.2 before reaching a flat plate 12 arranged in the illumination beam path (Fig. 6). The flat plate 12 is transparent to the illumination radiation but refractive. It can be pivoted about at least one, and advantageously about two, axes perpendicular to each other and to the optical axis oA. The pivoting movement can be initiated as a result of a control command from the control unit 11. By means of the flat plate 12, the illumination radiation can be adjusted in the beam path before it reaches the deflection device 2. In the exemplary embodiment, this is designed as a pair of sliding lenses. The focal length of the first optical element 2.1 can be changed in a controlled manner, thus fulfilling the function of a collimator.In the exemplary embodiment, a collimator is also formed by the axially displaceable optical lens 4.2 in conjunction with the first optical lens 4.1. In practice, the illustrated embodiment proves suitable, in which only one of the optical elements of the deflection device 2, namely the plano-concave lens 2.2, is laterally displaced, and the first optical element 2.1, shaped as a converging lens, is adaptive with respect to its focal length (symbolized by fΔ). This embodiment is less complex, and the required installation space is also reduced. The optical transfer function is: The term ei γ·x describes the beam deflection, with the term ei β·(x2+y2) describes a defocus, with By shifting the diverging lens by the distance x0, the beam deflection δ can be controlled, while the collimation (β) can be adjusted by varying the focal length of the first optical element 2.1. Both beam deflection and collimation can be adjusted independently of each other. After passing through the deflection device 2, the illumination radiation travels via the tube lens 5, color splitter 6, and objective 7 to the sample 8. A collected detection radiation returns to the color splitter 6 and passes through it. As in all embodiments, a filter 13 can optionally be present in the further detection beam path D. The tube lens 5 can, for example, be held together with other tube lenses 5 in a changer 14. The desired tube lens 5 can be introduced into the detection beam path D by actuating the changer 14. From the selected tube lens 5, the detection radiation is directed towards the detector 10. In a further embodiment of the illumination device 1 or the microscope M according to the invention, both the first optical element 2.1 and the second optical element 2.2 can be adaptive, i.e., their respective focal lengths (fadapt1 and fadapt2, respectively; abbreviated as fΔ in the figures) can be adjusted. Furthermore, both optical elements 2.1 and 2.2 can be displaced diametrically opposite to each other and perpendicularly with respect to the optical axis oA by a distance x0 (for the diametrical displacement, see also Fig. 2). This configuration offers the advantage that a defocus term (collimation) can also be generated, meaning that in addition to the deflection angle δ, the collimation of the illumination light can also be controlled. Furthermore, it is advantageous that the displacement x0 is set only once, for example, to define the operating point. Subsequently, only the focal lengths of the adaptive lenses are used. The optical transfer function for this configuration is: The term ei δ·x again describes the beam deflection, with the term ei β·x2 describes a defocus, with It should be noted that in equation (7) both a converging and a diverging adaptive lens is assumed, which is embodied by the different sign of the two terms in the transfer function. The equations show that, in addition to a displacement x0, both the beam deflection and the collimation can now be adjusted within a certain range by varying the focal lengths fadapt_1 and fadapt_2 of the two adaptive lenses 2.1 and 2.2. Two equations with two unknowns are given: δ = fkt(fadapt_1, fadapt_2) and β = fkt(fadapt_1, fadapt_2), which should provide a unique solution for fadapt_1 and fadapt_2. The solution for fadapt_1 and fadapt_2 becomes simple if either δ = 0 or β = 0, leading to fadapt_1 = -fadapt_2 or fadapt_1 = fadapt_2, respectively, thus simplifying the adjustment of the respective adaptive focal lengths. A dynamic variation of δ and / or β is also conceivable. In a third embodiment of an illumination device 1 of a microscope M according to the invention, the illumination radiation from a light source 3, for example a laser light source, reaches an optical grating 15 (Fig. 7) arranged in front of the plane plate 12. This grating divides the illumination radiation into beams of different diffraction orders, of which the zeroth and the ± first diffraction orders are considered below. The grid 15 can optionally be swung out of the lighting beam path B and, if necessary, swung or slid back in. This corresponds to the ability to switch the grid 15 between an activated and a neutral state. The second optical element 2.2 of the deflection device 2 is adjustable both laterally and with respect to its focal length. It can be used to position a spot in the projection plane PB or entrance pupil EP both laterally and axially. Depending on whether it is active or neutral, the grating 15 allows not only oblique illumination of the sample 8 but also the generation of an oblique illumination pattern by directing the diffraction orders into the entrance pupil EP in such a way that an illumination pattern is generated as a result of interference. Figure 8 shows a fourth embodiment of the invention. Unlike the third embodiment shown in Figure 7, the second optical element 2.2 is not only adjustable in terms of its focal length fadapt_2, but also axially adjustable. This allows the distance between the first optical element 2.1 and the second optical element 2.2 to be set. The beams of different diffraction orders generated by the action of the grating 15 (see also Figure 9) can be varied with respect to their lateral positions by axially positioning the second optical element 2.2. The adjustable focal length fadapt_2 serves to collimate and axially position the beams in the projection plane PB and the entrance pupil EP, respectively. For generating SIM illumination in a wide field, none of the optical elements 2.1, 2.2 are laterally adjusted. The beams of different diffraction orders are directed at different positions into the projection plane PB or the entrance pupil EP and are arranged rotationally symmetrically there. If the diffraction orders in the pupil plane PB are not centered on the optical axis oA, the misalignment can also be corrected using the described deflection device 2. In the sample space following the projection plane PB and the lens 7, i.e. in the area of ​​sample 8 and sample plane 9, an illumination pattern is generated as a result of interference. If the axial distance between the two optical elements 2.1, 2.2 is changed, aberrations can occur. These can be corrected, for example, with a variable phase plate, with at least one adjustable lens 4 and / or an SLM. In a further embodiment, the second optical element 2.2 can also be laterally adjustable. Together with the ability to control the grating 15, such a microscope M allows selection and switching between operating states of oblique illumination, oblique illumination with an illumination pattern, and wide-field illumination with an illumination pattern. A possible distribution of beams of different diffraction orders in the entrance pupil EP is shown by way of example in Fig. 9. A beam of the zeroth diffraction order is positioned in a spot (without pattern filling) on ​​the optical axis oA, while in each quadrant, a beam of the first diffraction order (shown filled with a stripe pattern) is positioned in a spot at the same radial distance to the optical axis oA. With an illumination device 1, for example according to Fig. 8, the radial positions of the spots of the first diffraction order can be shifted radially (see arrows) to a different position by changing the axial distance between the two optical elements 2.1, 2.2 (shown by way of example with a dot pattern). In this way, the TIRF-SIM illumination can be optimized. In all the aforementioned embodiments of the invention, the controller 11 can additionally control existing drives, for example, of the planar plate 12, the grid 15, and / or the changer 14. If the grid 15 is implemented, for example, by an SLM, the controller 11 can switch the grid 15 between different states. It is also possible that at least one variable lens is designed to adjust other Zernike orders in addition to defocus. Such a design can be used to correct aberrations in the plane conjugate to the intermediate image plane (ZB). Reference sign 1 Illumination device 2 Deflection device 2.1 First optical element 2.2 Second optical element 3 Light source 4 Optical lens 4.1 First lens 4.2 Second lens 5 Tube lens 6 Color splitter 7 Objective 8 Sample 9 Sample plane 10 Detector, camera 11 Control 12 Planar plate (wobbly plate) 13 Filter 14 Tube lens changer 15 Grating B Illumination beam path D Detection beam path δ Deflection angle EP Entrance pupil, Objective pupil fΔ Adaptive focal length M Microscope oA Optical axis PB Pupil plane, Projection plane ε Illumination angle x0 Travel ZB Intermediate image plane QUOTES INCLUDED IN THE DESCRIPTION This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature US 9,915,815 B2

[0005] DE 102 299 35 B4

[0005] US 2004 / 001253 A1

[0005] US 2014 / 0104680 A1

[0005] DE 102 589 45 A1

[0005] DE 10 2008 284 490 A1

[0005]

Claims

Illumination device (1) for illuminating a sample (8) with an illumination radiation along an illumination beam path (B), wherein an adjustable optical deflection device (2) is arranged in the illumination beam path (B) in or near an intermediate image plane (ZB), characterized in that the deflection device (2) is designed as a combination of at least one first and one second optical element (2.1, 2.2) in the form of optical lenses or optical wedges, of which at least one of the optical elements (2.1, 2.2) is adjustable with respect to its rotational position, its axial position and / or its lateral position, with respect to the optical axis (oA) of the illumination beam path (B), relative to the other optical element (2.1, 2.2).2) is adjustable, wherein, by the action of the deflecting device (2), at least one beam of the illumination radiation is or can be positioned in a projection plane (PB) extending perpendicular to the illumination beam path (B) in a targeted lateral position to the optical axis (oA), wherein the projection plane (PB) can optionally be used as an entrance pupil (EP) of a lens (7), and a control (11) is provided by means of which the deflecting device (2) can be controlled. Lighting device (1) according to claim 1 , characterized in that a lens (7) is provided whose entrance pupil (EP) lies in the projection plane (PB). Lighting device (1) according to claim 1 or 2, characterized in that in a first alternative as a combination of optical lenses an Abatscher wedge or a pair of sliding lenses or in a second alternative as a combination of optical wedges a pair of rotating wedges are provided. Lighting device (1) according to one of the preceding claims, characterized in that a collimator is arranged upstream of the deflection device (2), by the action of which an axial positioning of the beams, i.e. parallel to the optical axis (oA), can be set. Lighting device (1) according to one of the preceding claims, characterized in that the first and second optical element (2.1, 2.2) are movable in a controlled rotation or translation. Lighting device (1) according to claim 3, first alternative, characterized in that a pair of sliding lenses comprises a plano-convex optical lens as the first optical element (2.1) and a plano-concave optical lens as the second optical element (2.2). Lighting device (1) according to claim 6, characterized in that at least one of the two optical elements (2.1, 2.2) is designed as an adaptive lens and its respective focal length is adjustable. Lighting device (1) according to one of the preceding claims, characterized in that a planar plate (12) which is pivotable about at least one axis of rotation perpendicular to the optical axis (oA) is provided upstream of the deflecting device (2) in the lighting beam path (B), which is transparent but refracting for at least wavelength ranges of the lighting radiation. Lighting device (1) according to one of the preceding claims, characterized in that an optical grating (15) is arranged upstream of the deflection device (2), by the action of which the illumination radiation is divided into a plurality of beams of different diffraction orders and the beams can be positioned in the projection plane (PB) by the action of the deflection device (2). Microscope (M) comprising a lighting device (1) according to any one of claims 1 to 9. Microscope (M), in particular a wide-field microscope, comprising an illumination device (1) according to claims 1 to 9 for selectively illuminating a sample (8) with an illumination radiation at least in the first operating mode of oblique illumination and in the second operating mode of structured illumination, wherein an optical grating (15) can optionally be inserted into and removed from the illumination beam path (B), and in the first operating state the optical grating (15) is removed from the illumination beam path (B) and the illumination device (1) is configured according to one of claims 1 to 8; and in the second operating state the optical grating (15) is inserted into the illumination beam path (B) and the illumination device (1) is configured according to one of claims 1 to 9.

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