DEVICES FOR VERIFYING THE LATERAL AND AXIAL CONFOCALITANCE OF A SCANNING AND UNCANNING MICROSCOPE ASSEMBLY

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The invention relates to a scanning and descanning microscope assembly with a checking device for checking the confocality of the microscope assembly and to an auxiliary device for checking the confocality of a scanning and descanning microscope assembly.

[0002] The scanning and descanning microscope assembly comprises a light source providing illumination light, optics focusing the illumination light along an optical axis into a focus region, a detector detecting light from the focus region, and a scanner scanning laterally to the optical axis. The scanner is arranged between the light source and the detector on the one hand, and the focus region on the other. In such a scanning and descanning microscope assembly, a precise confocal arrangement of a detection aperture of the detector relative to the focus region into which the illumination light from the light source is focused is important for optimally measuring the light induced by the illumination light in the focus region. A precise confocal arrangement of the detection aperture is achieved when the detection aperture is arranged concentrically with the focus region of the illumination light with respect to its imaging into the focus region. STATE OF THE ART

[0003] To check the confocality of a scanning and descanning microscope assembly comprising a light source providing illumination light, an optical system focusing the illumination light into a focus in a focal plane, a detector detecting light from the focus and having a detection aperture arranged confocally to the focus, and a scanner between the light source and the detector on the one hand and the focal plane on the other, it is known from WO 2020 / 254303 A1 to scan an auxiliary detection aperture of an auxiliary detector arranged in the focal plane by adjusting the scanner with the focus of the illumination light. A first intensity distribution of the illumination light registered by the auxiliary detector is recorded across various scanner positions.Furthermore, by adjusting the scanner with auxiliary light emerging through an auxiliary emission aperture of an auxiliary light source arranged concentrically to the detection aperture in the focal plane, the detection aperture of the detector is scanned. A second intensity distribution of the auxiliary light registered by the detector is recorded across the scanner positions. At least one difference between the first intensity distribution and the second intensity distribution across the various scanner positions is then used as a measure of the confocality error of the scanning and descanning microscope assembly.

[0004] As an alternative to scanning the detection auxiliary aperture of the auxiliary detector with the focus of the illumination light, an auxiliary detection aperture of a separate auxiliary detector arranged concentrically to the emission aperture of the light source can be scanned by adjusting the scanner with the auxiliary light emerging through the auxiliary emission aperture of the auxiliary light source arranged in the focal plane in order to capture the first intensity distribution with the aid of the separate auxiliary detector. Furthermore, as an alternative to scanning the detection aperture of the detector with the auxiliary light from the auxiliary light source, the auxiliary detection aperture of the auxiliary detector arranged in the focal plane can be scanned by adjusting the scanner with further auxiliary light emerging through an auxiliary emission aperture of a separate auxiliary light source arranged concentrically to the detection aperture in order to capture the second intensity distribution with the aid of the auxiliary detector.

[0005] The difference of particular interest between the first intensity distribution and the second intensity distribution is a positional difference between the maxima or centroids of the two intensity distributions. To achieve or maintain the desired confocality, any positional difference is compensated by a real or virtual relative displacement of the detector's detection aperture relative to the light source.

[0006] The focal plane in which at least the detection auxiliary aperture or the emission auxiliary aperture is arranged can be an intermediate image plane of a laser scanning microscope comprising the scanning and descanning microscope assembly and an objective, or can be arranged in a branch of a main beam path of such a laser scanning microscope.

[0007] From US 2013 / 0 123 759 A1, a surface tracking and motion compensation system for a surgical tool is known. The system includes a surgical tool comprising a handpiece and a movable component, as well as a drive assembly. The drive assembly connects the movable component to the handpiece such that the movable component can be moved in an axial direction relative to the handpiece by the drive device. The system also includes an optical detection system comprising an optical fiber attached to the movable component, one end of the optical fiber being at a fixed distance from a distal portion of the movable component. The optical detection system is configured to output a signal for determining a distance of the distal portion of the movable component to a target during a surgical procedure. OBJECT OF THE INVENTION

[0008] The invention is based on the object of fundamentally improving the checking of the confocality of a scanning and descanning microscope assembly known from WO 2020 / 254303 A1. SOLUTION

[0009] The object of the invention is achieved by a scanning and descanning microscope assembly having the features of independent patent claim 1 and by means of an auxiliary device for checking the confocality of a scanning and descanning microscope assembly having the features of patent claim 12. Patent claims 2 to 11 are directed to preferred embodiments of the microscope assembly according to the invention and a laser scanning microscope with such an inventive microscope assembly. Patent claims 13 to 15 relate to preferred embodiments of the auxiliary device according to the invention. DESCRIPTION OF THE INVENTION

[0010] The present invention extends the verification of the confocality of a scanning and descanning microscope assembly known from WO 2020 / 254303 A1 by a verification of the axial confocality of the scanning and descanning microscope assembly. A special auxiliary detector and / or a special auxiliary light source providing auxiliary light are used. The auxiliary detector has an auxiliary detection aperture that can be moved into a plurality of auxiliary detection aperture positions spaced apart from one another in the direction of the optical axis, or a plurality of auxiliary detection apertures in a plurality of auxiliary detection aperture positions spaced apart from one another in the direction of the optical axis and laterally to the optical axis.The auxiliary light source has an auxiliary emission aperture which can be moved into a plurality of auxiliary emission aperture positions spaced apart from one another in the direction of the optical axis, or a plurality of auxiliary emission apertures in a plurality of auxiliary emission aperture positions spaced apart from one another in the direction of the optical axis and laterally to the optical axis, from which the auxiliary light emerges.

[0011] If the detection auxiliary aperture and / or the emission auxiliary aperture are movable, this does not require continuous movement. Rather, it is crucial that the detection or emission auxiliary aperture positions, which are spaced apart along the optical axis, can be precisely moved repeatedly.

[0012] The auxiliary detector and / or the auxiliary light source are arranged in a focus area of ​​a focusing optics of the scanning and descanning microscope assembly. When both the auxiliary detector and the auxiliary light source are arranged in the focus area, the auxiliary detection aperture and the auxiliary emission aperture, or the auxiliary detection apertures and the auxiliary emission apertures, are arranged concentrically in pairs. This implies that either both the auxiliary detection aperture and the auxiliary emission aperture are movable, or that multiple auxiliary detection apertures and multiple auxiliary emission apertures are present.

[0013] The scanning and descanning microscope assembly, the confocality of which is to be checked, comprises a first light source that provides first illumination light, a focusing optic that focuses the first illumination light along an optical axis into a focus area, a first detector that has a first detection aperture and detects first light from the focus area, and a scanner that scans laterally to the optical axis and that is arranged between the first light source and the first detector on the one hand and the focus area on the other hand.

[0014] The detector can be a point detector, whose detection aperture is defined by the active area of ​​its light-sensitive element or a pinhole arranged in front of it. However, the detector can also be an array detector with multiple light-sensitive elements. In this case, its detection aperture can be considered either the active area of ​​a central light-sensitive element, the total active areas of a central group of light-sensitive elements, or the total active areas of all light-sensitive elements. In principle, each auxiliary detector can also be designed either as a point detector or as an array detector, although a point detector is generally sufficient.

[0015] To check both the lateral and axial confocality of the scanning and descanning microscope assembly, the auxiliary detector and / or the auxiliary light source arranged in the focus area are used as follows.

[0016] By adjusting the scanner, the detection auxiliary aperture of the auxiliary detector, or the multiple detection auxiliary apertures of the auxiliary detector, are scanned with the focused first illumination light in the multiple detection auxiliary aperture positions. In this process, first intensity distributions of the first illumination light registered by the auxiliary detector are recorded across different scanner positions.

[0017] Alternatively, the first intensity distributions can be detected by scanning a first further detection auxiliary aperture of a first further auxiliary detector, arranged concentrically to a first emission aperture of the first light source, by adjusting the scanner with the auxiliary light emerging through the auxiliary emission aperture or the multiple auxiliary emission apertures of the auxiliary light source in the multiple auxiliary emission aperture positions. The first intensity distributions are then those of the auxiliary light and are detected by the first further auxiliary detector across the various positions of the scanner. An auxiliary emission aperture of the first and any other light source or auxiliary light source is understood here to mean the cross-sectional area from which the illumination light emerges.Furthermore, if the respective light source provides collimated light, each beam cross section of the collimated light can be regarded as an emission aperture and can be used for the purposes of the present invention.

[0018] Furthermore, the first detection aperture of the first detector is scanned by adjusting the scanner with the auxiliary light emerging through the auxiliary emission aperture or the multiple auxiliary emission apertures of the auxiliary light source in the multiple auxiliary emission aperture positions. Second intensity distributions of the auxiliary light registered by the detector are recorded across the various scanner positions.

[0019] Alternatively, the second intensity distributions can be acquired by scanning the detection auxiliary aperture or the multiple detection auxiliary apertures of the auxiliary detector in the multiple detection auxiliary aperture positions by adjusting the scanner with a first additional auxiliary light emitted through a first additional emission auxiliary aperture of a first additional auxiliary light source arranged concentrically to the first detection aperture. The second intensity distributions are those of the first additional auxiliary light and are acquired with the auxiliary detector across the scanner positions.

[0020] If multiple detection auxiliary apertures and / or multiple emission auxiliary apertures are present that are spaced apart laterally to the optical axis, their lateral spacing should separate the multiple detection or emission auxiliary apertures sufficiently during their respective scanning that the first and second intensity distributions are obtained separately or at least separable. However, the lateral spacing should not be much greater than necessary for this purpose.

[0021] The intensity distributions are compared with one another. In this case, a focus position of a focus of the focused first illumination light of the first light source in the focus region in the direction of the optical axis is detected from differences between the first intensity distributions; and a position of an image of the first detection aperture of the first detector in the focus region in the direction of the optical axis is detected from differences between the second intensity distributions. For these purposes, it is advantageous if the diameters of the detection and emission auxiliary apertures are approximately the same size as or smaller than the diameter of the focus of the focused first illumination light in the focus region or the diameter of the image of the first detection aperture in the focus region.

[0022] If the focus position and the image position match, axial confocality of the scanning and descanning microscope assemblies is present. However, the relevant axial confocality is only present if the focus position of the focus of the focused first illumination light of the first light source and the image position of the image of the first detection aperture of the first detector coincide in an object focus area on an object side of an objective in the direction of the optical axis. If the wavelengths of the first illumination light and the first light detected by the first detector differ, this does not necessarily mean, for example due to wavelength-dependent properties of the objective, that the focus position and the image position also coincide in another focus area, i.e., in the focus area in which the auxiliary detector and / or the auxiliary light source are arranged.

[0023] If the focus position and the image position do not coincide in the relevant object focus area, the focus position and the image position can be aligned by a real and / or virtual relative displacement of the first detection aperture of the first detector relative to the first light source in the object focus area. With respect to any focus area, deviations of the focus position from a target focus position and of the image position from a target image position can be detected from the differences between the first intensity distributions or the differences between the second intensity distributions. These deviations can then be compensated by real and / or virtual displacements of the first detection aperture of the first detector and / or the first light source.The target focus position and the target image position are to be determined such that the focus position of the focus of the focused first illumination light of the first light source and the image position of the image of the first detection aperture of the first detector coincide in an object focus area on an object side of an objective in the direction of the optical axis. In virtual displacements, it is not the first detection aperture of the first detector and / or the first light source itself that are displaced, but rather optical elements that preferably selectively influence the first light on its path to the first detection aperture or the first illumination light on its path from the first light source that are displaced or otherwise modified.

[0024] The relevant differences between the first intensity distributions and the second intensity distributions, from which the focus position or image position is inferred, depend in particular on the areas of the two-dimensional intensity distributions. These are, for example, the smallest areas that account for a certain percentage of the intensity, or the areas within which the intensity exceeds a certain limit, e.g., a percentage of the mean or maximum intensity of the intensity distribution. The focus position or image position of interest is found where the area of ​​the respective intensity distribution has its minimum along the optical axis. This minimum can be at one or between several of the detection or emission auxiliary aperture positions.If the beam shape of the respective illumination light, auxiliary light, and / or additional auxiliary light in the area of ​​the respective detector, auxiliary detector, and / or additional auxiliary detector is known—for example, if you know that it is a Gaussian beam with a specific beam waist and Rayleigh length—two different detection or emission auxiliary aperture positions along the optical axis are sufficient. To reliably locate the minimum even without detailed prior knowledge, three different detection or emission auxiliary aperture positions along the optical axis, which are also distributed around the focus position or image position, are advantageous. In addition to the areas of the intensity distributions, their shapes can also be considered when considering their differences.In order to increase the informative value of the shapes of the intensity distributions, a distortion such as astigmatism can be deliberately introduced into the focusing optics, as is generally known to the person skilled in the art.

[0025] To verify the lateral confocality, the teaching of WO 2020 / 254303 A1 can be used. Thus, by comparing one of the first intensity distributions with one of the second intensity distributions, in particular by comparing the positions of their maxima, centers, and / or centroids, conclusions can be drawn about the lateral confocality or the degree of any deviation from the desired lateral confocality of the scanning and descanning microscope assembly. This conclusion is particularly simple if the compared first and second intensity distributions correspond to the same detection or emission auxiliary aperture positions. Otherwise, existing differences between the detection or emission auxiliary aperture positions underlying the first and second intensity distributions must be taken into account.

[0026] The auxiliary detector and / or the auxiliary light source, i.e., the auxiliary detection aperture positions and / or the auxiliary emission aperture positions, can be arranged around an object plane of a microscope beam path of a laser scanning microscope comprising the scanning and descanning microscope assembly and an objective. This arrangement in the area of ​​the object plane is only possible temporarily, because the samples to be examined with the laser scanning microscope must be arranged there. Furthermore, the auxiliary detection and / or emission apertures and the distances between their auxiliary detection and / or emission aperture positions must be microscopically small for their arrangement in the area of ​​the object plane.Even when arranging the detection auxiliary aperture positions and / or the emission auxiliary aperture positions around an intermediate image plane of the microscope beam path, it may be necessary to remove the auxiliary detector and / or the auxiliary light source for the actual use of the laser scanning microscope. However, if the detection auxiliary aperture positions and / or the emission auxiliary aperture positions are arranged in a branch of a main beam path of the microscope beam path around an intermediate image plane, the auxiliary detector and / or the auxiliary light source can be arranged stationary there.

[0027] In order to be able to reliably determine the focus position or the image position from the first or second intensity distributions, it is advantageous if the detection auxiliary aperture positions and / or the emission auxiliary aperture positions are each distributed along the optical axis over a distance of 0.5 mm to 10 mm. This absolute specification of the distance relates to the arrangement of the detection auxiliary apertures or emission auxiliary apertures around an intermediate image plane of the microscope beam path, i.e. on the side of the objective facing away from the object side. Relative to a magnification M proportional to the objective magnification between the object focus area on the object side of the objective and the intermediate image plane, around which the detection auxiliary aperture positions and / or the emission auxiliary aperture positions are arranged, the distance over which the detection auxiliary aperture positions orEmission auxiliary aperture positions are preferably distributed along the optical axis, in a range of 0.2 µm x M 2< to 1 µm x M 2< .

[0028] Advantageously, the plurality of detection auxiliary apertures of the auxiliary detector and / or the plurality of emission auxiliary apertures of the auxiliary light source have identical dimensions and shapes transverse to the optical axis, i.e., when viewed along the optical axis. Only then are the first and second intensity distributions directly comparable with one another. Preferably, the detection auxiliary aperture(s) of the auxiliary detector and the emission auxiliary aperture(s) of the light source are not only concentric in pairs, but also coincident. To achieve this, at least one photoelectric component can be used both as part of the auxiliary light source and as part of the auxiliary detector. This photoelectric component can be, for example, a light-emitting diode, a superluminescent diode, a laser diode, or a photodiode.Alternatively, the auxiliary detection aperture(s) of the auxiliary detector and / or the auxiliary emission aperture(s) of the auxiliary light source can be formed with at least one end cross-section of a light guide. This light guide can then be branched to the auxiliary light source on the one hand and the auxiliary detector on the other. This branching can be realized, for example, with a free-beam beam splitter, a fiber beam splitter, or a circulator.

[0029] In a preferred embodiment, the auxiliary detection apertures of the auxiliary detector and / or the auxiliary emission apertures of the auxiliary light source are formed with holes in different layers of a layered structure or at different depths of a plate. On a rear side of the layered structure or the plate, the beam path to the auxiliary light source and the auxiliary detector can be branched.

[0030] The scanning and descanning microscope assembly can further comprise a second light source providing second illumination light, wherein the focusing optics focus the second illumination light along the optical axis into the focus region. If the auxiliary detection aperture or the multiple auxiliary detection apertures of the auxiliary detector are then scanned in the multiple auxiliary detection aperture positions by adjusting the scanner with the focused second illumination light, wherein further first intensity distributions of the second illumination light registered by the auxiliary detector are detected across the various scanner positions, a further focal position of a focus of the focused second illumination light of the second light source in the focus region in the direction of the optical axis can be detected from differences between the further first intensity distributions.This allows the axial confocality of the scanning and descanning microscope assembly to be checked with respect to the second illumination light. This second illumination light can, for example, be fluorescence-preventing light, more specifically, so-called STED light.

[0031] Alternatively, the further first intensity distributions can be detected by scanning a second further detection auxiliary aperture of a second further auxiliary detector, arranged concentrically to a second emission aperture of the second light source, by adjusting the scanner with the auxiliary light emerging through the emission auxiliary aperture or the multiple emission auxiliary apertures of the auxiliary light source in the multiple emission auxiliary aperture positions. Then, the further first intensity distributions are those of the auxiliary light and are detected by the separate second auxiliary detector across the various positions of the scanner.

[0032] The scanning and descanning microscope assembly can further comprise a second detector that detects light from the focus area and has a second detection aperture. This second light can, for example, have a different wavelength and originate from different emitters than the first light during normal use of the scanning and descanning microscope assembly. To check the confocality of the scanning and descanning microscope assembly with respect to the second detector, the second detection aperture of the second detector can be scanned by adjusting the scanner with the auxiliary light emerging through the auxiliary emission aperture or the multiple auxiliary emission apertures of the auxiliary light source in the multiple auxiliary emission aperture positions, wherein further second intensity distributions of the auxiliary light registered by the second detector are recorded across the scanner positions.From differences between the further second intensity distributions, a further image position of an image of the second detection aperture of the second detector in the focus area in the direction of the optical axis can then be detected, which allows conclusions to be drawn about the axial confocality.

[0033] Alternatively, the further second intensity distributions can be detected by scanning the detection auxiliary aperture or the multiple detection auxiliary apertures of the auxiliary detector in the multiple detection auxiliary aperture positions by adjusting the scanner with a second auxiliary light emitted through a second auxiliary emission aperture of a second auxiliary light source arranged concentrically to the second detection aperture. The further second intensity distributions are those of the second auxiliary light and are detected with the auxiliary detector across the scanner positions.

[0034] The scanning and descanning microscope assembly according to the invention comprises, in addition to the first light source, the focusing optics, the first detector, and the scanner, the auxiliary detector and / or the auxiliary light source defined above, as well as a checking device configured to detect the first and second intensity distributions. The comparison of the intensity distributions can also be performed by the checking device. Furthermore, the checking device can be configured not only to detect deviations of the focus position from a target focus position and the image position from a target image position, but also to compensate for them by real and / or virtual relative displacements of the first detection aperture of the first detector relative to the first light source.The target focus position and the target image position are preferably again set such that the focus position of the focus of the focused first illumination light of the first light source and the image position of the image of the first detection aperture of the detector coincide in an object focus region on an object side of an objective of a laser scanning microscope comprising the scanning and descanning microscope assembly according to the invention in the direction of the optical axis in order to establish the axial confocality.

[0035] To establish lateral confocality, the inspection device can apply the teaching of WO 2020 / 254303 A1.

[0036] A laser scanning microscope according to the invention comprises, in addition to the microscope assembly according to the invention, an objective, wherein the detection auxiliary aperture positions and / or the emission auxiliary aperture positions are arranged around an intermediate image plane. This intermediate image plane can be an intermediate image plane of a microscope beam path comprising the scanning and descanning microscope assembly and the objective, or can be located in a branch of a main beam path of such a microscope beam path.

[0037] An auxiliary device according to the invention for checking the confocality of a scanning and descanning microscope assembly has a connector designed for connection in a defined relative position to the scanning and descanning microscope assembly. The optical axis, along which the detection auxiliary aperture positions and the emission auxiliary aperture positions of the auxiliary detector defined above and the auxiliary light source defined above are arranged as parts of the auxiliary device, runs in a fixed orientation to the connector. The auxiliary detector and the auxiliary light source can be designed in detail as already explained above. The connector of the auxiliary device can be a standardized connector and, for example, fit an objective connector of the scanning and descanning microscope assembly.

[0038] Advantageous further developments of the invention emerge from the patent claims, the description and the drawings.

[0039] The advantages of features and combinations of several features mentioned in the description are merely exemplary and can be effective alternatively or cumulatively, without the advantages necessarily having to be achieved by embodiments according to the invention.

[0040] The number of features mentioned in the claims and the description is to be understood as meaning that exactly this number or a greater number than the stated number is present, without the need for the explicit use of the adverb "at least." For example, if a lens is mentioned, this is to be understood as meaning that exactly one lens, two lenses, or more lenses are present. The features mentioned in the claims may be supplemented by further features or may be the only features present in the subject matter of the respective claim.

[0041] The reference signs contained in the patent claims do not represent a limitation of the scope of the subject-matter protected by the patent claims. They serve solely to make the patent claims easier to understand. BRIEF DESCRIPTION OF THE CHARACTERS

[0042] In the following, the invention is further explained and described with reference to preferred embodiments shown in the figures. Fig. 1 shows a laser scanning microscope according to the invention with a microscope assembly according to the invention. Fig. 2 shows a first embodiment of an auxiliary device of the laser scanning microscope according to Fig. 1 . Fig. 3 shows a second embodiment of the auxiliary device of the laser scanning microscope according to Fig. 1 . Fig. 4 illustrates a combination of a first detector and a first further auxiliary light source in a modification of the laser scanning microscope according to Fig. 1 . Fig. 5 illustrates the combination of a first light source and a first additional auxiliary detector in another modification of the laser scanning microscope according to Fig. 1 . Fig. 6shows a concrete embodiment of an auxiliary device according to the invention for use in the laser scanning microscope according to Fig. 1 . Fig. 7 shows a detail of the auxiliary device according to Fig. 6 . Fig. 8 shows an alternative version of the detail according to Fig. 7 ; and Fig. 9 shows another alternative version of the detail according to Fig. 7 . FIGURE DESCRIPTION

[0043] The Fig. 1 The schematically illustrated laser scanning microscope 1 comprises an objective lens 2 and a microscope assembly 3, the components of which are outlined by a dashed line. The microscope assembly 3 includes a first light source 4, in particular in the form of a laser 5, for providing first illumination light 23. Furthermore, the microscope assembly 3 includes an optical system 27 that focuses the illumination light 23 into a focus 47 in a focus area 7 around a focal plane 6. Fig. 1The focal plane 6 is shown with a dashed line. As part of the focusing optics 27, Fig. 1only lens 28 is shown. In addition, the microscope assembly 3 includes a detector 8 for first light 9 from the focus area 7. In front of the actual detector 8, i.e., its light-sensitive area, there is a pinhole 10 and another optics 29, which focuses the light 9 to be detected by the detector 8 onto the opening of the pinhole 10. The opening of the pinhole 10 defines a detection aperture 11 of the detector 8, which, when the microscope assembly 3 is correctly adjusted, is arranged confocally to the focus 47 in the focus area 7. A scanner 12 of the microscope assembly 3 is arranged between the light source 4 and the detector 8 with the pinhole 10 on the one hand and the focal plane 6 on the other. The lens 28 of the optics 27 is located on the side of the scanner 12 facing the light source 4 and the detector 8.The scanner 12 comprises two rotating mirrors 13, 14 for each lateral direction in which the scanner 12 is intended to scan a sample 15 arranged in front of the objective 2 with the illumination light 23 from the light source 4. Each rotating mirror 13, 14 is assigned a rotary drive 44. The scanner 12 is used not only to scan the sample 15 with the illumination light 23, but also to descan the light 9 which is caused by the illumination light 23 in the sample 15 so that this light is spatially selectively detected by the detector 8. This depends on an exact confocality of the microscope assembly 3, i.e. an exact coincidence of the focus of the illumination light 23 focused onto the sample 15 and an image of the detection aperture 11 of the detector 8 onto the sample 15. This confocality is shown in the diagram in . Fig. 1drawn focal plane 6 is checked. This focal plane 6 lies in a branch 16 from the main beam path 17 of the laser scanning microscope 1 and corresponds to an intermediate image plane in the main beam path 17. A tube lens 33 is arranged in the main beam path 17 between the scanner 12 and the objective 2. The branch 16 branches off from the main beam path 17 at the last rotating mirror 14 of the scanner 12 in front of the objective 2. If the branch is activated via this last rotating mirror 14, the other rotating mirrors 13, 14 can be used to scan an auxiliary device 18 arranged in the branch 16. The auxiliary device 18 is located in a fixed position around the focal plane 6 relative to the other components of the microscope assembly 3 and can be reached by controlling the scanner 12 with the illumination light 23.

[0044] The beam path of the first illumination light 23 from the first light source 4 and the beam path of the first light 9 to be detected by the first detector 8 are separated by means of a beam splitter 26. Another beam splitter 36 separates the beam path of the second illumination light 35 from a second light source 34, which is also typically designed as a laser 5, from the beam path of the light 9. A beam shaper 32 is arranged in the beam path of the second light 35. The beam shaper 32 can be provided to create an intensity distribution of the second illumination light 35 with a central intensity minimum in the focus area 7, as is common for fluorescence-preventing light, in particular STED light, to improve resolution.A further beam splitter 31 separates a beam path of the second light 30 to be detected from the beam path of the first light 9 to be detected, wherein the first light 9 and the second light 30 can differ, in particular, in their wavelength. The second light 30 is detected by a second detector 38, whose second detection aperture 41 is defined by a further pinhole 40.

[0045] Furthermore, Fig. 1 a testing device 37 is schematically shown, which is connected to the light sources 4, 34, the detectors 8, 38, the scanner 12 and the auxiliary device 18 and with the aid of these connections carries out the checking of the confocality of the scanning and descanning microscope assembly 3.

[0046] Except for the additional second light source 34, the additional second detector 38, the reproduction of the test device 37 and the only schematic representation of the auxiliary device 18 corresponds Fig. 1the Fig. 5 WO 2020 / 254303 A1. The other embodiments of the scanning and descanning microscope assembly 3 disclosed in WO 2020 / 254303 A1 and one of the laser scanning microscopes 1 constructed therewith can also be upgraded and used with an auxiliary device 18, as will be explained in more detail below. In this regard, reference is made to the disclosure of scanning and descanning microscope assemblies 3 and laser scanning microscopes 1 in WO 2020 / 254303 A1.

[0047] Fig. 2 schematically illustrates an embodiment of the auxiliary device 18. The focal plane 6 and the focal area 7 as well as an optical axis 39 of the branch 16 are shown, along which the illumination light 23 is incident on the auxiliary device 18. The auxiliary device 18 here has a photoelectric component 19, which can be used both as an auxiliary light source 20 and as an auxiliary detector 24. Fig. 2Three pairs of congruent auxiliary emission apertures 21 of the auxiliary light source 20 and auxiliary detection apertures 25 of the auxiliary detector 24 are formed by diaphragms (not shown in detail). The auxiliary emission apertures 21 and the auxiliary detection apertures 25 are spaced apart from one another in the direction of the optical axis 39 and laterally thereto. Fig. 2 The unspecified connection 73 serves to connect the auxiliary device 18 in a defined relative position to the other components of the microscope assembly 3.

[0048] After a first step A, in which the emission auxiliary apertures 21 and the detection auxiliary apertures 25 are arranged in the axially and laterally spaced emission and detection auxiliary aperture positions in the focus area 7, in a subsequent step B, by adjusting the scanner 12, the detection auxiliary apertures 25 of the auxiliary detector 24 are scanned with the focused first illumination light 23 in order to detect first intensity distributions of the first illumination light 23 registered by the auxiliary detector 24 across different positions of the scanner 12.

[0049] Then, in a subsequent step C, the photoelectric component 19 is activated as an auxiliary light source 20, and the first detection aperture 11 of the first detector 8 is scanned with the auxiliary light 22 emerging from the auxiliary emission apertures 21 while adjusting the scanner 12. In this process, second intensity distributions of the auxiliary light 22 registered by the first detector 8 are recorded across the position of the scanner 12. During a subsequent comparison of the light intensity distributions in a step D, a focus position of the focus 47 of the focused first illumination light 23 of the first light source 4 in the focus area 7 in the direction of the optical axis 39 is recorded from differences between the first intensity distributions. An image position of an image of the first detection aperture 11 of the first detector 8 in the focus area 7 in the direction of the optical axis 39 is recorded from differences between the second intensity distributions.The differences depend in particular on the areas of the intensity distributions. From the determined focus position and the determined image position, conclusions can be drawn about the axial confocality of the microscope assembly 3, whereby the focus position and the image position do not have to coincide at the auxiliary device 18, but rather in the sample 15. Due to wavelength-dependent properties of the objective 2 and different wavelengths of the illumination light 23 and the auxiliary light 22, this may require that the focus position and the image position in the focus area 7 on the auxiliary device 18 assume desired focus and desired image positions spaced apart in the direction of the optical axis 39. The lateral confocality of the microscope assembly 3 is checked by comparing the first and second intensity distributions, particularly in the pairs corresponding to a pair of auxiliary emission aperture 21 and auxiliary detection aperture 25.This is achieved when maxima, central or centroidal points of the first and second light intensity distributions coincide along the focal plane 6 in the focal area 7.

[0050] Fig. 3 shows an alternative embodiment of the auxiliary device 18 with only one emission auxiliary aperture 21 and a detection auxiliary aperture 25 coinciding therewith in the focus area 7 around the focus plane 6. Both apertures 21, 25 are formed by an end cross-section 46 of a light guide 40. The light guide 40 branches via a circulator 41 to a photodiode 42 serving as an auxiliary detector 24 and a laser diode 43 serving as an auxiliary light source 20. With the aid of a displacement device 45, on which the Fig. 3 The end cross-section 46 can be moved into various emission auxiliary aperture positions or detection auxiliary aperture positions. In each of these positions, the scanner 12 can be used according to Fig. 1 a first light intensity distribution and a second light intensity distribution are detected. The displacement device 45 can be specially designed to move the end cross-section 46 into a certain number of discrete emission auxiliary aperture positions or detection auxiliary aperture positions, for example, into three such discrete positions. The circulator according to Fig. 3 is just one example of a beam splitter to branch the light guide 40 to the auxiliary detector 24 and the auxiliary light source 20.

[0051] Instead of providing both an auxiliary detector 24 and an auxiliary light source 20 in the auxiliary device 18, it can also be designed as an auxiliary detector 24 with several auxiliary detection apertures 25. Then, to detect the second intensity distributions, the first detector 8 and possibly also the second detector 38 can be combined with a further auxiliary light source 48, as shown in Fig. 4is outlined. The first detection aperture 11 is formed by a light guide 49, which branches off via a circulator 50 to a photodiode 51 serving as the detector 8 and to a laser diode 52 serving as the further auxiliary light source 48. The further emission auxiliary aperture 53 of the further auxiliary light source 48 is identical to the first detection aperture 11 of the first detector 8. With further light emerging from the further emission auxiliary aperture 53 from the further auxiliary light source 48, the detection auxiliary apertures 25 or the movable detection auxiliary aperture 25 can be scanned in the various detection auxiliary aperture positions in order to detect the second intensity distributions with the auxiliary detector 24.

[0052] Accordingly, the auxiliary device 18 can alternatively have only one auxiliary light source 20 with several emission auxiliary aperture positions of its emission auxiliary aperture(s) 21. Then, to detect the first intensity distributions, the first light source 4 and accordingly also the second light source 34 can be combined with a further auxiliary detector 54, as shown in Fig. 5is illustrated. The free end cross-section of a light guide 55, which branches via a circulator 56 to a laser diode 57 serving as the first light source 4 and a photodiode 58 serving as the further auxiliary detector 54, forms both a first emission aperture 59 of the first light source 4 and a further detection auxiliary aperture 60 of the further auxiliary detector 54. To detect the first intensity distributions with the further auxiliary detector 54, this further detection auxiliary aperture 60 is scanned with auxiliary light from the emission auxiliary apertures 21 or the emission auxiliary aperture 21 in the various emission auxiliary aperture positions of the auxiliary light source 20. Also in the Fig. 4 and 5The light guide 59 or 55 and its branching to the photodiodes 51 or 58 and the laser diodes 52 or 57 are merely exemplary. Further possible implementations of the detector 8 and the further auxiliary light source 48 with a matching detection aperture 11 and a further auxiliary emission aperture 53, or of the light source 4 and the further auxiliary detector 54 with a matching emission aperture 59 and a further auxiliary detection aperture 60, are accessible to the person skilled in the art without inventive effort.

[0053] Regarding the direction of the emitted or registered light, the diodes 42, 43, 51, 52, 57 and 58 are in the Figures 3 to 5 not represented with the usual symbols, but with self-selected symbols.

[0054] At the Fig. 6In the specific embodiment of the auxiliary device 18 shown, various optical components are arranged on a base plate 61 serving as a connection 73 and in a base body 62, which are screwed together with screws 63. The emission auxiliary apertures 21 and detection auxiliary apertures 25 are formed in pairs by holes 64 in foils 65, as in connection with Fig. 7 will be explained in more detail below. Behind the holes 64, a beam splitter 66 branches the beam path to the photodiode 42 serving as the auxiliary detector 24 and the laser diode 43 serving as the auxiliary light source 20. The beam splitter 66 is designed here as a 50 / 50 beam splitter.

[0055] Fig. 7shows how the holes 64 forming the auxiliary emission apertures 21 and the auxiliary detection apertures 25 are provided in the three foils 65, which are spanned at intervals along the optical axis 39 between edge regions 67 of the base body 63. Precisely one such hole 64 is provided in each of the foils 65. The holes 64 are spaced laterally from the optical axis 39. Along the optical axis 39, larger holes 68 are made in the other foils 65, aligned with the holes 64, which have no influence on the respective auxiliary emission aperture 21 or auxiliary detection aperture 25.

[0056] Fig. 8 shows an alternative design of the holes 64 at different emission or detection auxiliary aperture positions along and across the optical axis 39. Here, the holes 64 are the smallest diameter areas of conical or double-conical bores 69 in a plate 70.

[0057] When the emission auxiliary apertures 21 and the detection auxiliary apertures 25 are formed through the holes 64 according to Fig. 9 Blind holes 71 are drilled into the plate 70 until it is removed to thin remnants 72, which are located in different positions along the optical axis 39 and transversely thereto. One of the holes 64 is then centrally inserted into each of these thin remnants 72. LIST OF REFERENCE SYMBOLS

[0058] 1 Laser scanning microscope 2 Lens 3 Microscope assembly 4 First light source 5 Laser 6 Focus plane 7 Focus area 8 First detector 9 First light 10 Pinhole 11 First detection aperture 12 Scanner 13 Rotating mirror 14 Rotating mirror 15 Sample 16 Branch 17 Main beam path 18 Auxiliary device 19 Photoelectric component 20 Auxiliary light source 21 Emission auxiliary aperture 22 Auxiliary light 23 First illumination light 24 Auxiliary detector 25 Detection auxiliary aperture 26 Beam splitter 27 Optics 28 Lens 29 Additional optics 30 Second light 31 Beam splitter 32 Beam shaper 33 Tube lens 34 Second light source 35 Second illumination light 36 Beam splitter 37 Test device 38Second detector 39Optical axis 40Light guide 41Circulator 42Photodiode 43Laser diode 44Rotary drive 45Traversing device 46End cross section 47Focus 48Further auxiliary light source 49Light guide 50Circulator 51Photodiode 52Laser diode 53Further auxiliary emission aperture 54Further auxiliary detector 55Light guide 56Circulator 57Laser diode 58Photodiode 59First emission aperture 60FurtherDetection auxiliary aperture 61Base plate 62Base body 63Screw 64Hole 65Foil 66Beam splitter 67Edge area 68Hole 69Conical hole 70Perforated plate 71Blind hole 72Thin remainder 73Connection

Claims

1. Scanning and descanning microscope component group (3) comprising (a) a first light source (4) providing first illumination light (23); (b) focusing optics (27) focusing the first illumination light (23) along an optical axis (39) into a focal area (7); (c) a first detector (8) having a first detection aperture (11) and detecting first light coming out of the focal area (7); (d) a scanner (12) arranged between the first light source (4) and the first detector (8) , on its one side, and the focal area (7), on its other side, and scanning laterally with respect to the optical axis (39); (A) an auxiliary detector (24) arranged or to be arranged in the focal area (7), the first auxiliary detector comprising - an auxiliary detection aperture (25) movable into a plurality of auxiliary detection aperture positions arranged at distances in direction of the optical axis (39) or - a plurality of auxiliary detection apertures (25) in a plurality of auxiliary detection aperture positions arranged at distances in direction of the optical axis (39) and laterally with respect to the optical axis (39), and / or an auxiliary light source (20) arranged or to be arranged in the focal area (7) and providing auxiliary light (22), the auxiliary light source comprising - an auxiliary emission aperture (21) movable into in a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis (39) or - a plurality of auxiliary emission apertures (21) in a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis (39) and laterally with respect to the optical axis (39), through which the auxiliary light (22) emerges, wherein, if both the auxiliary detector (24) and the auxiliary light source (20) are arranged in the focal area (7), the auxiliary detection aperture(s) (25) and the auxiliary emission aperture(s) (21) are arranged concentrically in pairs; and a checking device (37); (B) wherein the checking device (37) is configured for scanning the auxiliary detection aperture (25) or the plurality of auxiliary detection apertures (25) of the auxiliary detector (24) in the plurality of auxiliary detection aperture positions with the focused first illumination light ( 23) by setting the scanner (12), wherein the checking device (37) registers first intensity distributions of the first illumination light (23) detected by the auxiliary detector (24) over different settings of the scanner (12), or a first further auxiliary detector (54) having a first auxiliary detection aperture (60) arranged concentrically with respect to a first emission aperture (59) of the first light source (4) is provided and wherein the checking device (37) is configured for scanning the first auxiliary detection aperture (60) with the auxiliary light (22) that emerges through the auxiliary emission aperture (21) or the plurality of auxiliary emission apertures of the auxiliary light source (20) in the plurality of auxiliary emission aperture positions by setting the scanner (12), wherein the checking device (37) registers first intensity distributions of the auxiliary light (22) detected by the first further auxiliary detector (54) over different settings of the scanner (12); and (C) wherein the checking device (37) is configured for scanning the first detection aperture (11) of the first detector (8) with the auxiliary light (22) that emerges through the auxiliary emission aperture (21) or the plurality of auxiliary emission apertures (21) of the auxiliary light source (20) in the plurality of auxiliary emission aperture positions by setting the scanner (12), wherein the checking device (37) registers second intensity distributions of the auxiliary light (22) detected by the first detector (8) over the settings of the scanner (12), or a first further auxiliary light source (48) providing first further auxiliary light and having a first further auxiliary emission aperture (53), which is arranged concentrically with respect to the first detection aperture (11) and through which the first further auxiliary light emerges, is provided, and wherein the checking device (37) is configured for scanning the auxiliary detection aperture (25) or the plurality of auxiliary detection apertures (25) of the auxiliary detector (24) in the plurality of auxiliary detection aperture positions with the first further auxiliary light that emerges through the first further auxiliary emission aperture (53) by setting the scanner (12), wherein the checking device (37) registers second intensity distributions of the first further auxiliary light detected by the first auxiliary detector (24) over the settings of the scanner (12).

2. Scanning and descanning microscope component group (3) of claim 1, characterized in that the checking device (37) is configured for comparing the first intensity distributions and for determining a focus position of a focus (47) of the focused first illumination light (23) of the first light source (4) in the focal area (7) in direction of the optical axis (39) from differences between the first intensity distributions, and an image position of an image of the first detection aperture (11) of the first detector (8) in the focal area (7) in direction of the optical axis (39) from differences between the second intensity distributions.

3. Scanning and descanning microscope component group (3) of any of the claims 1 and 2, characterized in that plurality of auxiliary detection apertures (25) of the auxiliary detector (24) and / or the plurality of auxiliary emission apertures (21) of the auxiliary light source (20) have same shapes and dimensions orthogonal to the optical axis (39).

4. Scanning and descanning microscope component group (3) of any of the claims 1 to 3, characterized in that the auxiliary detection aperture(s) (25) of the auxiliary detector (24) and the auxiliary emission aperture(s) (21) of the auxiliary light source (20) are congruent in pairs.

5. Scanning and descanning microscope component group (3) of claim 3 or 4, characterized in that at least one photoelectric component (19) is both part of the auxiliary light source (20) and of the auxiliary detector (24), wherein the photoelectric component (19) optionally is a light emitting diode, a super luminescence diode, a laser diode or a photo diode.

6. Scanning and descanning microscope component group (3) of claim 3 or 4, characterized in - that the auxiliary detection aperture(s) (25) of the auxiliary detector (24) and the auxiliary emission aperture(s) (21) of the auxiliary light source (20) are provided by an least one end cross section (39) of an optical fiber (40), wherein the optical fiber (40) is optionally branched towards the auxiliary light source (20) and the auxiliary detector (24) by means of an open beam beam splitter, a fiber beam splitter or a circulator (41), or - that the auxiliary detection aperture(s) (25) of the auxiliary detector (24) and the auxiliary emission aperture(s) (21) of the auxiliary light source (20) are provided by holes (64) in different layers of a layered structure or in different depths of a plate (70), wherein a beam path on the back side of the respective layered structure or of the plate (70) is optionally branched towards the auxiliary light source (20) and the auxiliary detector (24).

7. Scanning and descanning microscope component group (3) of any of the claims 1 to 6, characterized in - that the scanning and descanning microscope component group (3) comprises a second light source (34) providing second illumination light (35), wherein the focusing optics (27) focus the second illumination light (35) along the optical axis (39) in the focal area (7); - that the checking device (37) is configured for scanning the auxiliary detection aperture (25) or the plurality of auxiliary detection apertures (25) of the auxiliary detector (24) in the plurality of auxiliary detection aperture positions with the focused second illumination light (35) by setting the scanner (12), wherein the checking device (37) registers further first intensity distributions of the second illumination light (35) detected by the auxiliary detector (24) over different settings of the scanner (12), or a second further auxiliary detector having a second further auxiliary detection aperture arranged concentrically with respect to a second emission aperture of the second light source (34) is provided, wherein the checking device (37) is configured for scanning the second further auxiliary detection aperture with the auxiliary light (22) that emerges through the auxiliary emission aperture (21) or the plurality of emission apertures (21) of the auxiliary light source (20) in the plurality of auxiliary emission aperture positions by setting the scanner (12), wherein the checking device (37) registers further first intensity distributions of the auxiliary light (22) detected by the second further auxiliary detector over different settings of the scanner (12); and that the checking device (37) is configured for determining a further focus position of a focus of the focused second illumination light of the second light source in the focal area in direction of the optical axis from differences between the further first intensity distributions.

8. Scanning and descanning microscope component group (3) of claim 7, characterized in that the second illumination light (35) provided by the second light source (34) is fluorescence inhibiting light.

9. Scanning and descanning microscope component group (3) of any of the claims 1 to 8, characterized in - that the scanning and descanning microscope component group (3) comprises a second detector (38) detecting second light (30) coming out of the focal area (7) and having a second detection aperture (41); - that the checking device (37) is configured for scanning the second detection aperture (41) of the second detector (38) with the auxiliary light (22) that emerges through the auxiliary emission aperture (21) or the plurality of auxiliary emission apertures (21) of the auxiliary light source (20) in the plurality of auxiliary emission aperture positions by setting the scanner (12), wherein the checking device (37) registers further second intensity distributions of the auxiliary light (22) detected by the second detector (38) over the settings of the scanner (12), or a second further auxiliary light source providing second further auxiliary light and having a second further auxiliary emission aperture, which is arranged concentrically with respect to the second detection aperture (41) and through which the second further auxiliary light emerges, is provided, wherein the checking device (37) is configured for scanning the auxiliary detecting aperture (25) or the plurality of auxiliary detection apertures (25) of the auxiliary detector (24) in the plurality of auxiliary detection aperture positions with the second further auxiliary light from the second further auxiliary light source by setting the scanner (12), wherein the checking device (37) registers further second intensity distributions of the second further auxiliary light detected by the first auxiliary detector (24) over the settings of the scanner (12); and wherein the checking device (37) is configured for determining a further image position of an image of the second detection aperture (41) of the second detector in the focal area (7) in direction of the optical axis (39) from differences between the further second intensity distributions.

10. Laser scanning microscope (1) comprising - an objective and - a microscope component group (3) of any of the claims 1 to 9, - wherein the auxiliary detection aperture positions and / or the auxiliary emission aperture positions are arranged around an intermediate image plane of a microscope beam path including the scanning and descanning microscope component group (3) and the objective (2) or - the auxiliary detection aperture positions and / or the auxiliary emission aperture positions are arranged around an intermediate image plane in a branch (16) that branches off a main microscope beam path (17) of a microscope beam path including the scanning and descanning microscope component group (3) and the objective (2).

11. Laser scanning microscope (1) of claim 10, characterized in that the auxiliary detection aperture positions and / or the auxiliary emission aperture positions are each distributed along the optical axis (39) over a distance of 0,5 mm to 10 mm and / or over a distance of 0.2 µm x M2 to 1 µm x M2, wherein M is a magnification between an object focal area on an object side of the objective (2) and the intermediate image plane.

12. Auxiliary apparatus (18) for checking the confocality of a scanning and descanning microscope component group (3), the auxiliary apparatus (18) comprising - a connector (73) configured for connecting the auxiliary apparatus (18) in a defined relative position to The scanning and descanning microscope component group (3); - an optical axis (39) running at a fixed orientation with respect to the connector (73); - an auxiliary detector (24) comprising - an auxiliary detection aperture (25) moveable with respect to the connector (73) into a plurality of auxiliary detection aperture positions that are arranged at distances in direction of the optical axis (39), or - a plurality of auxiliary detection apertures (25) in a plurality of auxiliary detection aperture positions that are arranged at distances in direction of the optical axis (39) and laterally with respect to the optical axis (39); and an auxiliary light source (20) providing auxiliary light (22) and comprising - an auxiliary emission aperture (21) moveable with respect to the connector (73) into a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis (39), or - a plurality of auxiliary emission apertures (21) in a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis (39) and laterally with respect to the optical axis (39).

13. Auxiliary apparatus (18) of claim 12, characterized in that the auxiliary detection aperture(s) (25) and the auxiliary emission aperture(s) (21) - are arranged concentrically in pairs and / or - are distributed along the optical axis (39) over a distance of 0,5 mm to 10 mm and / or - have same shapes and dimensions orthogonal to the optical axis (39).

14. Auxiliary apparatus (18) of claim 12 or 13, characterized in that at least one photoelectric component (19) is both part of the auxiliary light source (20) and part of the auxiliary detector (24), wherein the photoelectric component (19) optionally is a light emitting diode, a super luminescence diode, a laser diode or a photo diode.

15. Auxiliary apparatus (18) of claim 12 or 13, characterized in - that the auxiliary detection aperture(s) (25) of the auxiliary detector (24) and the auxiliary emission aperture(s) (21) of the auxiliary light source (20) are provided by an least one end cross section (39) of an optical fiber (40), wherein the optical fiber (40) is optionally branched towards the auxiliary light source (20) and the auxiliary detector (24) by means of an open beam beam splitter, a fiber beam splitter or a circulator (41), or - that the auxiliary detection aperture(s) (25) of the auxiliary detector (24) and the auxiliary emission aperture(s) (21) of the auxiliary light source (20) are provided by holes (64) in different layers of a layered structure or in different depths of a plate (70), wherein a beam path on the back side of the respective layered structure or of the plate (70) is optionally branched towards the auxiliary light source (20) and the auxiliary detector (24).