Device and method for confocal observation of a sample

Abstract

Devices and methods for confocal observation of a scattering, preferably fluorescent, sample with a microscope, wherein the device comprises at least one disc-like body, preferably made of glass, wherein the at least one disc-like body has on a first side a plurality of micro-optical elements and on a second side a correspondingly arranged pinhole pattern formed by a plurality of pinholes, wherein the plurality of micro-optical elements arranged on the first side have a convex, dichroic-coated front surface, wherein, for excitation of the sample, the disc is illuminated on its side facing the tube lens with collimated light, which is reflected back to the tube lens by the plurality of curved, dichroic-coated surfaces on the disc in such a way that a corresponding excitation point pattern is formed in the object plane of the microscope, whichAfter passing through the tube lens and the multitude of curved, dichroic-coated surfaces, it can be spatially filtered and detected.

Description

Technical area

[0001] The present disclosure relates to the field of microscopy. In particular, the present invention relates to devices and methods for spatially (confocally) filtered imaging of fluorescent samples. background

[0002] In all areas of microscopy, efforts are being made to increase the optical resolution and contrast of images. One approach, particularly used in fluorescence microscopy, uses a spatially filtering pinhole to block out-of-focus light during image formation. The light reflected or emitted by the sample is filtered through a pinhole aperture located precisely in the image plane of the microscope system. This predominantly transmits light from a specific focal plane, while blocking light from outside this focal plane. This filtering reduces interfering background signals and improves the contrast ratio and image quality. The result is what is known as confocal imaging.

[0003] A variant of this confocal imaging technique, which spatially filters excitation and emission light, relies on the arrangement of movable pinholes on a rotating disk. A perforated mask rotates around a central axis, and all points of the object being observed are illuminated through the holes for as equal a duration as possible. Such a rotating disk is generally referred to as a Nipkow disk. Nipkow microscope systems are fast and gentle on samples, but suffer from poor light yield if the excitation beam path only uses the light that passes through the pinholes without first being focused through them. This problem can be overcome by focusing the excitation light onto the pinholes. This typically involves a second rotating disk carrying a corresponding array of microlenses.This disc is positioned in front of the pinhole disc in the illumination beam path, so that the pinhole plane coincides with the focal plane of the microlenses, and the focal points generated by the microlenses fall precisely into the pinhole openings. When both discs rotate around a common axis, many (usually > 1,000) simultaneously illuminated points are obtained on the sample. Due to the rotation, these points illuminate the entire area of ​​the sample seen by the detector, and the emission light generated there is captured with confocal filtering through the pinholes.

[0004] Such spinning disk systems, i.e. confocal microscope systems based on the Nipkow principle, are enjoying increasing popularity in live cell microscopy.

[0005] Some significant disadvantages of such methods, in which excitation and emission beams must pass through the same pinholes, result from the merging and separation of the excitation and emission beams, which must occur in the finite optical space between the microlens and pinhole disk. In addition, the two disks must be precisely aligned.

[0006] DE 10 2015 112 960 B3 describes a device for the confocal observation of a sample with a rotating disk and a plurality of pinholes for confocal filtering of the emitted light. In this device, unlike the prior art (EP 0 539 691 A2, DE 10 2007 009 551 B3), collimated excitation light does not come from behind the disk, as seen from the microscope, where it is formed into illumination spots by micro-optical elements and passes through the pinholes toward the microscope, but rather from the front! According to DE 10 2015 112 960 B3, the rotating disk is illuminated with collimated light from the direction of the microscope, reflected by the disk, and formed into excitation spots, which produce the desired excitation pattern in the sample that rotates with the disk.Beam shaping and reflection occur on two different surfaces, (i) a micro-optically curved (therefore refracting) first surface, which allows the collimated light to converge, and (ii) a flat, mirrored surface, which reflects the converging light beams back. After passing through the curved surface (i) for the second time, the beams converge in front of the disk to form excitation spots, which the microscope images onto the sample. To ensure that the resulting emission image does not also end up in front of the disk, where there is no possibility of confocal filtering, DE 10 2015 112 960 B3 places the pinholes for confocal filtering as openings in the flat, reflecting surface (ii) on the disk and separates or combines the excitation and emission beams.with each other using two planar dichroic mirrors in such a way that the first, after passing through a path-length shifting arrangement, is recombined with the emission beam so that the image of the emitted spots falls exactly into the conjugated pinholes. Separation and combination for path-length compensation are achieved in DE 10 2015 112 960 B3 using two planar dichroic reflecting surfaces. However, this has the disadvantage that the required separation and subsequent combination of the excitation and emission beams involves the highly critical adjustment of two reflection angles, and the length of the detour must also be adjusted very precisely.

[0007] DE 10 2022 108 448 B3 describes a device for microscopic imaging of fluorescent samples with increased resolution using a disk equipped with micro-optical elements and pinholes, rotating around a central axis, wherein the reference light and the emission light pass through the disk at different radial distances from the central axis.

[0008] US 11,543,640 B2 and JP 7 415 674 B2 contain helpful information that facilitates understanding of the background of the present invention. Summary of the Invention

[0009] The object of the present invention is to at least partially overcome the disadvantages known in the prior art. The above object is achieved by a structured pane according to claim 1, a device according to claim 6, and a method according to claim 10. Preferred embodiments are the subject of the respective subclaims.

[0010] In one aspect, the present invention is directed to a structured disk for confocal observation of a sample, in particular a fluorescent sample, using an imaging optical system. According to the invention, the structured disk comprises at least one disk-like body, preferably formed from glass, wherein the at least one disk-like body has a plurality of micro-optical elements on a first side and a correspondingly arranged pinhole pattern formed by a plurality of pinholes on a second side, wherein the plurality of micro-optical elements arranged on the first side have a convex, dichroically coated front surface. According to the invention, the disk-like body can be formed from any material known to the person skilled in the art that has the corresponding optical properties or can be provided with them.Likewise, mixtures and combinations of different materials can also be used. The dichroic coating is determined according to the invention depending on the respective application and is to be selected such that the excitation light to be used is reflected, but the resulting emission light is transmitted. Accordingly, in preferred embodiments, the coating can be a long-pass coating in which excitation light is reflected and emission light is transmitted. Alternatively, the coating can be a multi-band coating in which several wavelength ranges are reflected and / or absorbed. Preferably, the micro-optical elements and the pinholes are each correlated in pairs, i.e. arranged around their respective central axis.

[0011] In one embodiment of the present invention, the distance between the first and the second side of the disc can be selected such that light which is inverse to divergent light resulting from reflection of collimated light at the convex micro-optical elements of the first side is focused into the respective pinholes after transmission of the dichroic coating.

[0012] In one embodiment of the structured disk according to the present invention, the convexly curved, dichroically coated surfaces can be cast in a medium, wherein the medium preferably has the same refractive index as the plurality of micro-optical elements. The convexly curved, dichroically coated surfaces can be cast in such a way that a non-curved surface arranged parallel to the disk-shaped body is formed, whereby the plane of the virtual illumination spots coincides with the plane of the pinhole mask.

[0013] In a further embodiment of the structured pane according to the present invention, the plurality of convexly curved dichroic coated surfaces on the first side may be opposed by a second arrangement of convexly curved surfaces on the opposite side.

[0014] In a further embodiment of the structured disk according to the present invention, the disk may comprise a first disk-shaped body and a second disk-shaped body, wherein the plurality of micro-optical elements are arranged on the first disk-shaped body and the plurality of pinholes are arranged on a second disk-shaped body in a correlated manner.

[0015] In a further embodiment of the structured disc according to the present invention, the structured disc may comprise a second disc-shaped body on which a plurality of conjugated diverging lenses are arranged, each diverging lens being arranged with a micro-optical element and a pinhole corresponding around respective central axes.

[0016] In a further aspect, the present invention is directed to a device for confocal observation of a fluorescent sample with a microscope, wherein the device comprises at least one structured disk, as described above, which is rotatable about a central axis. The structured disk is arranged between a tube lens and a detector of the microscope such that the plurality of micro-optical elements face the tube lens. To excite the sample, the disk is illuminated on its side facing the tube lens with collimated light, which, due to the plurality of curved, dichroic-coated surfaces on the disk, creates a virtual point pattern behind these surfaces. The rays are reflected back to the tube lens in such a way that, in interaction with the objective of the microscope, a corresponding excitation point pattern is created in the object plane of the microscope.The excitation point pattern creates a fluorescent point pattern in the sample. After passing backward through the objective and tube lens, it re-enters the multitude of curved, dichroic-coated surfaces where the excitation light was previously reflected. However, due to the dichroic properties of the reflective surfaces, it is now transmitted. Because the transmitted beams of light combine to form focal points in front of the plane of the virtual excitation point pattern due to the refractive power of the curved surfaces, they are spatially (confocally) filtered in this plane by a correspondingly arranged pinhole pattern. According to the invention, this pinhole plane can be imaged onto a detector, in particular a camera, using optics. A detector within the meaning of the present invention is any means for detecting the emitted light and can accordingly be a camera, a light-sensitive chip, or even the human eye.

[0017] In one embodiment of the device according to the present invention, the device further comprises a coupling mirror arranged centrally in the focal plane of the tube lens. The coupling mirror can preferably be aligned in the image beam path such that light focused onto the mirror by a focusing element, after passing through the tube lens, strikes the disk and the plurality of micro-optical elements as collimated excitation light perpendicularly and is reflected from there back to the tube lens. In a preferred embodiment, the coupling mirror can be arranged on a glass element that transmits both excitation light and emission light, and can be arranged with this glass element at least partially in the beam path.

[0018] In another embodiment, a large mirror can be used, preferably at an angle of 45° to the objective axis, with a small coupling hole on the beam axis in or near the objective pupil. This mirror can deflect the emission and excitation light (by 90°). In this embodiment, the excitation light can be focused through the coupling hole of the mirror, creating the required collimated light field on the disk.

[0019] In a further embodiment of the device according to the present invention, the focal plane of the tube lens may coincide with the back focal plane of the objective.

[0020] In a further embodiment of the device according to the present invention, the structured disk rotatable about a central axis can be replaced by a plane dichroic mirror, wherein the plane dichroic mirror is tilted by an angle (∂), whereby the reflected excitation light in the focal plane of the tube lens, which preferably coincides with the focal plane of the objective, is directed past the coupling mirror or the coupling hole and is formed by the objective into an oblique wide-field illumination of the sample.

[0021] In a further aspect, the present invention is directed to a method for confocal observation of a scattering, preferably fluorescent, sample using a microscope. The method comprises the steps of illuminating the sample and detecting confocally filtered emission or scattered light. Illuminating the sample comprises illuminating a plurality of curved, dichroic-coated surfaces on a structured disk arranged between a tube lens and a detector of a microscope, in particular a structured disk as described above, with collimated light. According to the invention, the structured disk is arranged between the tube lens and the detector of a microscope such that the plurality of curved, dichroic-coated surfaces face the tube lens.By illuminating the multitude of curved, dichroic-coated surfaces with collimated light, a virtual dot pattern is created behind these surfaces, and the rays are reflected back to the tube lens, creating a corresponding excitation dot pattern in the object plane of the microscope. The resulting excitation pattern, in turn, creates a fluorescent dot pattern, which, after passing backward through the tube lens, re-enters the multitude of curved, dichroic-coated surfaces where the excitation light was reflected. However, due to the dichroic properties of the reflective surfaces, it is now transmitted. Because the transmitted beams of light combine to form focal points in front of or in the plane of the virtual excitation dot pattern due to the refractive power of the curved surfaces, they are spatially (confocally) filtered in this plane by the appropriately arranged pinhole pattern. Brief description of the figures

[0022] The accompanying figures illustrate exemplary embodiments and applications of the present invention. Herein: Fig. 1a and Fig. 1b a device according to the invention; Fig. 2a and Fig. 2b embodiments according to the invention for increasing resolution; Fig. 3a and Fig. 3b the coupling of collimated excitation light into the device according to the invention; Fig. 4 a wide-field illumination according to the invention; Fig. 5a and Fig. 5b Embodiments of structured panes according to the invention; Fig. 6 a flowchart of the method according to the invention. Detailed description

[0023] The present invention will now be described in more detail with reference to the accompanying drawings. However, the invention may be embodied in many different forms and should not be construed as limited to the embodiments shown herein. It should be noted that the figures illustrate general features of the apparatus and methods utilized in the respective embodiments. However, these figures may not depict the exact structure or feature of a given embodiment. Furthermore, like reference numerals in the figures designate corresponding parts throughout the different views or embodiments.

[0024] The present invention as described in Fig. 1a and Fig. 1b, circumvents the disadvantage of DE 10 2015 112 960 B3 by involving a virtual path length 11. This reduces the required path length compensation until it disappears completely and avoids the need to separate and recombine the excitation and emission beams because a single, curved dichroic surface 3 is sufficient for this. Its focal point 6 is virtual in nature and is not located - as seen from the microscope - in front of, but behind the convex reflecting surfaces 3. The optical system thus becomes self-adjusting, the critical angular adjustment of the two dichroics is eliminated and the virtual (negative) focal length 11 during focus generation compensates for the required path length difference 12, in the case shown to a large extent. The remaining amount is precisely determined by the thickness of the glass substrate 4, i.e.adjustable by the distance between the microlens surface 3 refractive for the emission light and the pinhole mask 5 applied to the back of the glass substrate 2.

[0025] Fig. Figure 1a shows the microscope setup according to the invention, omitting the part that generates the collimated excitation light 1, which preferably strikes the disk perpendicularly, between the tube lens 8 and the disk 4. After reflection from the dichroic-coated surface 3 of the micro-optical elements of the structured disk, designed here as microlenses 2, the rays of the excitation point pattern 7 are formed, which appear to come from the virtual focus 6 of the convexly curved virtual surface 3 of the microlens 2. Due to the coating, the convexly curved surfaces 3 are convex mirrors for the excitation light. They are imaged by the tube lens 8 to infinity and by the objective 9 into the sample 10, where fluorescence excitation occurs. The resulting emission light 21 ( Fig. 1b) passes through the objective lens 9 and the tube lens 8 back to the dichroic-coated lens surface 3, which is transparent to the emission light and, as lens 2, exhibits refractive properties. Thus, the real focal points, with a path length difference of 12, lie in front of the virtual focal plane 6 of the dichroic mirror 3, in the pinhole plane 5. Due to the refractive power of the microlens 2, the numerical aperture (NA) of the spots in the pinhole plane 5 is increased by a factor of the path lengths 11 / (11-12), and the focal points are compressed accordingly, leading to a certain increase in resolution. If this is not desired, the refractive power of the microlens 2 can be eliminated by embedding the curved surface 3 in a medium that has the same refractive index as the microlens 2. This reduces the property to a micro-optical element with a curved, dichroic mirrored surface.

[0026] One possibility to maximize the resolution increase mentioned, ie up to a doubling of the original emission spot NA (NA 28 = 2x NA 27), consists in increasing the refractive power of the microlenses, as in Fig. 2a. The pinholes 26 are then transferred to a second disk 25 and precisely aligned with the microlenses 22 and 23 of the first disk 24. Confocal filtering then occurs at the already compressed spots.

[0027] Alternatively, as in Fig. As shown in Figure 2b, the NA 38 of the emission light cone 31 is subsequently virtually enlarged after passing through the pinholes 35 on the back of the disc 34, and the virtual spot size in the plane 37 is correspondingly reduced. For this purpose, a second disc 36 is paired behind the first disc 34 with a pattern of diverging lenses 33 conjugated to the pinholes 35 of the first disc 34. The two discs 34 and 36 can be separated by an air gap 39, but this can also be reduced to zero by cementing both discs 34 and 36 together.

[0028] In order to couple the collimated excitation light 41 into the microscope in such a way that it homogeneously illuminates the desired image field 42 on the disk 44 rotating about the axis 43, an intermediate image field 45 conjugated to the image field on the disk 42 is required, the emission NA of which is so low that the excitation light 41 generated therefrom with the aid of the tube lens 47 and the focusing element 48 has the desired collimation when it strikes the disk 44. An intermediate image field 45 with dimensions of 14 × 14 mm should emit light with an NA < 0.001, provided the focal lengths of the two tube lenses 47 and 48 are the same.

[0029] Fig. Figure 3a shows the most compact implementation: The excitation light 41 is coupled into a glass plate 50, which is centrally mirrored in the focal plane 46 of the focusing element 48, and the desired reflection angle 51 is adjusted. For a preferably selected telecentric arrangement of the entire illuminating and imaging system, the focal plane 46 of the focusing element 48 coincides with the rear focal plane 40 of the objective 59. If the focal plane of the objective 59 were located accessible outside the objective 59, the central mirroring 49 could concentrate on a very small spot that obstructs only a vanishingly small percentage of the pupil area.However, since the rear focal plane 40 of the objective 59 is usually located inside the objective 59, the inclined glass plate 50 must be mounted well in front of the objective pupil 40 and illuminated in such a way that the real focal point 146 of the tube lens 47 is located in front of the mirrored surface 49 and the virtual focal point is inside the objective 59, in its pupil plane 40, as in . Fig. 3b shown.

[0030] If the coupled light is used for telecentric illumination of the disk, a field of the same size as in the intermediate illumination plane 45 is illuminated there with the same degree of collimation, with the light beams reflected back toward the microscope having an NA that fills the objective pupil. The central obstruction caused by the coupling mirror in front of the pupil plane amounts to only a few percent of the total pupil area and, as long as it is precisely centered, has no image degradation effect!

[0031] To accommodate situations where the sample is not to be illuminated with rotating focus patterns, but rather—for example—with planar wide-field illumination, it is not sufficient in the present invention to simply remove the disk 44 from the beam, because then the collimated excitation light 41 would be wasted. In order to excite fluorescence in the sample 60, the excitation light 41 must be reflected back toward the tube lens 48 and objective 59. If the disk 44 were simply replaced by a flat dichroic mirror 63, the excitation light 41 would be reflected back onto the coupling mirror 49 and directed by it back to its starting point.However, it is possible to generate (oblique) wide-field illumination in the sample 60 by tilting a dichroic mirror by an angle (∂), whereby the reflected excitation light 61 in the focal plane 46 of the tube lens 48, which preferably coincides with the focal plane 40 of the objective 59, is directed past the coupling mirror 49 and is formed by the objective 59 into an oblique wide-field illumination of the sample 60 (. Fig. 4).

[0032] If the angle (∂) is chosen large enough, the process known as TIRF (Total Internal Reflection Fluorescence) can also be realized in this way. This requires the positioning of an illumination spot so far towards the edge of the pupil of a specially designed TIRF objective that the beam is totally reflected at the interface to the object and only excites layers in the sample close to the cover glass. If the dichroic mirror 63 is coupled to a tilting device 62, the "obliqueness" of the illumination can be adjusted up to the TIRF angle. If 62 is a two-axis tilting device, the sample can even be excited from different directions obliquely or in TIRF mode. Particularly good images are obtained if the spot 64 of the excitation beam 61 is guided in a circle around its central axis in the objective pupil 40 during the exposure of an image.

[0033] The described modification of the beam path to generate homogeneous wide-field illumination allows an examination of the sample, as used, for example, in the STORM or PALM method to further increase the resolution.

[0034] Another fluorescence technique that benefits from the described wide-field laser illumination is the rapid mapping of extended sample areas with a continuously moving sample. The sample is illuminated with short flashes of light while it is moved across the objective, and the pixel size, adjusted by binning, is adjusted to the remaining motion blur. A 40x objective, for example, has a pixel resolution of 100 nm. If a virtual resolution of 1 µm is desired, 10 × 10 pixels can be captured within and well-controlled images can be obtained even with an exposure time of 50 µs. To also limit the motion blur to 1 µm, the sample must not be moved faster than 20 mm / s. A field of 20 × 20 mm can thus be captured in just over 40 seconds with 4x virtual magnification.This makes it possible to begin an examination immediately with the desired high-resolution lens, a significant advantage over the current state of the art, especially if the desired lens is an immersion lens. Typically, the overview image is acquired with a low-magnification lens in stop-and-go mode, and only then can the user switch to the desired high-resolution lens.

[0035] The Fig. 5a and Fig. 5b each show embodiments of the structured pane according to the invention, as they are used, for example, in the methods and devices according to the invention. Fig. 5a shows a structured disk 20 formed from a glass body 4. On a first side of the disk 20, a plurality of micro-optical elements 2 are arranged, each of which has convexly curved, dichroically coated front surfaces 3 on its side facing away from the disk. The coating is selected depending on the respective application so that excitation light is reflected and emission light is transmitted. On the other side of the structured disk 20, a plurality of pinholes 5a are arranged in a so-called pinhole plane 5, wherein the number of pinholes 5a corresponds to the number of micro-optical elements and the pinholes and the micro-optical elements are each arranged correspondingly, i.e. aligned around a common central axis. Fig. 5b shows a further embodiment of a structured disc 30. The structured disc 30 essentially corresponds to the one shown in Fig. 5a, with the difference that the refractive power of the micro-optical elements 2 was made to disappear by embedding the curved surface 3 in a medium 9 having the same refractive index as the micro-optical elements 2.

[0036] Fig. Figure 6 shows a flowchart of the method 600 according to the invention for confocal observation of a fluorescent sample with a microscope. In a first step 610 of the method, a structured disk arranged between the tube lens and the detector of a microscope is illuminated with collimated light. The structured disk is a Fig. 5a and Fig.5b, wherein the disc is arranged in the microscope according to the invention such that the side with the micro-optical elements faces the tube lens and the side with the pinholes faces the detector. By illuminating the plurality of micro-optical elements with collimated light, a virtual point pattern is formed behind the plurality of curved, dichroic-coated surfaces 620. Due to the dichroic coating of the curved surfaces, the rays forming the virtual point pattern are reflected 630 toward the tube lens, thus forming an excitation point pattern in the object plane of the microscope 640, which in turn generates a fluorescent emission point pattern 650. In the subsequent step 660, this emission light passes both the tube lens and, due to the dichroic coating, the micro-optical elements, and is then confocally filtered through the pinholes 670.In a final step 680, the confocally filtered emission light can be detected by means of a suitable detector.

[0037] The scope of this disclosure includes all changes, substitutions, variations, alterations, and modifications to the embodiments described or illustrated herein that would be obvious to one skilled in the art. The scope of this disclosure is not limited to the embodiments described or illustrated herein. Moreover, although this disclosure describes and illustrates particular embodiments herein as including particular components, elements, features, functions, operations, or steps, any of these embodiments may include any combinations or permutations of any components, elements, features, functions, operations, or steps that would be obvious to one skilled in the art.Furthermore, a reference in the appended claims to a method, apparatus, or component of a device or system being adapted, arranged, capable, configured, enabled, operable, or ready to perform a particular function includes that device, system, or component, regardless of whether it or that particular function is enabled, turned on, or enabled, as long as that device, system, or component is adapted, arranged, capable, configured, enabled, operable, or ready to perform it. Furthermore, while this disclosure describes or illustrates certain embodiments as being advantageous, certain embodiments may provide none, some, or all of these advantages.

Claims

[1] Structured disc for confocal observation of a sample with an imaging optical system, comprising at least one disc-like body preferably formed from glass, wherein the at least one disc-like body has on a first side a plurality of micro-optical elements and on a second side a correspondingly arranged pinhole pattern formed by a plurality of pinholes, wherein the plurality of micro-optical elements arranged on the first side each have a convex, dichroic coated front surface. [2] Structured disc according to claim 1, wherein the distance between the first and second sides is selected such that light which is inverse to divergent light resulting from reflection of collimated light at the convex micro-optical elements of the first side is focused into the respective pinholes after transmission of the dichroic coating. [3] Structured disc according to claim 1 or 2, wherein the convexly curved, dichroic coated surfaces are cast in a medium having the same refractive index as the plurality of micro-optical elements, and thus have an uncurved surface arranged parallel to the disc-shaped body, whereby the plane of the virtual illumination spots coincides with the plane of the pinhole mask. [4] A structured pane according to claim 1 or 2, wherein the plurality of convexly curved dichroic coated surfaces on the first side is opposed by a second arrangement of convexly curved surfaces on the opposite side. [5] Structured disc according to claim 3, wherein the disc comprises a first disc-shaped body and a second disc-shaped body, wherein the plurality of micro-optical elements are arranged on the first disc-shaped body and the plurality of pinholes are arranged on a second disc-shaped body in a correlated manner. [6] Structured disc according to one of claims 1 to 3, wherein the disc comprises a second disc-shaped body on which a plurality of conjugated diverging lenses are arranged, each diverging lens being arranged with a micro-optical element and a pinhole corresponding around respective central axes. [7] Device for confocal observation of a fluorescent sample with a microscope, the device comprising: a structured disc according to one of claims 1 to 6, rotatable about a central axis, arranged between a tube lens and a detector of the microscope, which is illuminated with collimated light on the side facing the tube lens to excite the sample, which, due to the plurality of curved, dichroic-coated surfaces on the disc, creates a virtual point pattern behind these surfaces, and the rays are reflected back to the tube lens in such a way that a corresponding excitation point pattern is created in the object plane of the microscope, which generates a fluorescent point pattern that, after passing backward through the tube lens, hits the plurality of curved, dichroic-coated surfaces again where the excitation light had been reflected, but is now transmitted due to the dichroic property of the reflecting surfaces,and the distance between the first side of the disc and the second side of the disc is selected such that beams of the transmitted fluorescent dot pattern combine to form focal points in the plane of the pinholes and can be spatially (confocally) filtered by the pinhole pattern. [8] Device according to claim 7, wherein the device further comprises a coupling mirror arranged centrally in the focal plane of the tube lens, wherein the coupling mirror is aligned in the image beam path such that light focused on the mirror by a focusing element after passing the first tube lens strikes the plurality of micro-optical elements as collimated excitation light and is reflected from there back to the first tube lens. [9] Apparatus according to claim 8, wherein the focal plane of the tube lens coincides with the back focal plane of the objective. [10] Device according to claim 8 or 9, wherein the structured disc rotatable about a central axis is replaced by a plane dichroic mirror, wherein the plane dichroic mirror is tilted by an angle (∂), whereby the reflected excitation light in the focal plane of the tube lens, which preferably coincides with the focal plane of the objective, is directed past the coupling mirror and is formed by the objective into an oblique wide-field illumination of the sample. [11] A method for confocal observation of a fluorescent sample with a microscope, the method comprising: Illuminating a plurality of curved, dichroic mirrored surfaces on a structured disc arranged between a tube lens and a detector of a microscope according to one of claims 1 to 6 with collimated light, whereby a virtual point pattern is generated behind these surfaces and the rays are reflected back to the tube lens in such a way that a corresponding excitation point pattern is created in the object plane of the microscope, which generates a fluorescent point pattern which, after passing backwards through the tube lens, strikes the plurality of curved, dichroic mirrored surfaces again where the excitation light had been reflected, but is now transmitted due to the dichroic property of the reflecting surfaces, wherein the distance between the first side of the disc and the second side of the disc is selected such thatthat beams of the transmitted fluorescent dot pattern combine in the plane of the pinholes to form focal points and are spatially (confocally) filtered by the pinhole pattern; and , Detecting the confocally filtered fluorescent dot pattern with the detector. [12] The method according to claim 11, further comprising coupling collimated excitation light via a coupling mirror arranged centrally in the focal plane of the tube lens, wherein the coupling mirror is aligned in the image beam path such that light focused onto the mirror by a second tube lens, after passing through the first tube lens, strikes the plurality of curved, dichroic mirrored surfaces as collimated excitation light and is reflected from there back to the first tube lens.

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