REFERENCE SAMPLE FOR A MICROSCOPE AND METHOD FOR CALIBRATION AND REFERENCE

Description

[0001] The invention relates to a reference sample according to the preamble of independent claim 1 and uses of the reference sample.

[0002] Optical imaging systems, especially high-resolution microscopes, can be calibrated and / or adjusted using reference samples. Such reference samples are provided with two-dimensional structures in the form of test patterns that were created in a carrier material of the reference sample or are applied to the carrier material. These test patterns have substructures whose location on the reference sample, their dimensions, and their distances from one another, especially their lateral distances, are known in advance.

[0003] For example, reference samples in the form of a slide or cuvette, which are common in the field of light microscopy, are known. These are made of glass and feature two-dimensional test patterns (structures) that can be excited to emit fluorescent radiation. Based on the fluorescent radiation detected by the test patterns, the resolving power of the microscope configuration used can be evaluated, for example.

[0004] The excitation and emission of fluorescence radiation are based on doping the glass, particularly with so-called rare earth elements. Fluorescence centers generated in this way degenerate far less rapidly under excitation radiation and environmental conditions than protein-based fluorescent markers or inorganic marker molecules. However, even the doped glasses exhibit a sometimes significant decrease in emitted intensities upon repeated excitation (e.g., Royon, A. and Converset, N., 2017: Quality Control of Fluorescence Imaging Systems - A new tool for performance assessment and monitoring; Optik & Photonik 2 / 2017: 22-25).

[0005] A possible use of diamonds with defects as reference structures is generally addressed, for example, in the press release of the Max Planck Institute for Biophysical Chemistry dated February 25, 2009 (Anonymous: "Nano instead of carats - diamonds in the service of science", press release of the Max Planck Institute for Biophysical Chemistry, February 25, 2009).

[0006] The structures used as test samples have so far been introduced into the substrate using technical processes whose resolution corresponds to the resolution of the optical systems to be measured, calibrated, or adjusted, especially microscopes. Therefore, a metrological determination of the resolution of high-resolution microscopes is only possible to a very limited extent using the reference samples known from the state of the art.

[0007] The invention is therefore based on the object of proposing a reference sample that enables improved resolution measurements compared to the prior art. Furthermore, the invention is intended to create a way to enable comparative measurements on optical systems and imaging systems, in particular microscopes, that are not influenced by degradation of the luminescence intensity from the sample.

[0008] The object is achieved by the subject matter of the independent claims. Advantageous embodiments of the invention can be found in the dependent claims.

[0009] The reference sample is used to measure, calibrate, and / or adjust an optical imaging system such as a microscope, in particular a high-resolution microscope, and has at least one support structure made of a support material that can be excited to emit light (luminescence) at least in regions of its extent, i.e. at least in regions or sections. The support material also has at least one two- and / or three-dimensional structure, predetermined in terms of its dimensions and shape, consisting of a number of substructures. This two- and / or three-dimensional structure forms the test sample. The reference sample can have multiple structures, which can also differ from one another in terms of their dimensions and design. The structures are also referred to below as test samples.

[0010] According to the invention, the carrier material consists of diamond, which can be produced by technical means. Another embodiment of the invention uses silicon carbide (SiC) as the carrier material. To ensure that the carrier material can be excited to emit light, the carrier material is doped in or around the structured areas.

[0011] The support structure allows for repeated provision and use of the test samples and contains or carries the structuring. The support structure can, for example, be mounted on a slide commonly used in microscopy.

[0012] The respective partial structure is formed laterally and / or axially as a surface, as points, as a three-dimensional structure, and / or as a combination of at least two line segments. A lateral partial structure extends on or below a surface of the reference sample and substantially parallel to it, while an axial partial structure extends into the carrier material in a z-direction or obliquely to it. According to the invention, the axial partial structures comprise at least one pair of intersecting lines.

[0013] An optical imaging system, such as a microscope, is referred to as high-resolution if it can image structures smaller than the Abbe limit. The invention is explained below using one or more microscopes as examples.

[0014] Excitation for the emission of luminescence occurs in particular by means of excitation radiation of suitable wavelength and intensity.

[0015] In an advantageous embodiment of the reference sample, the support material is doped with nitrogen vacancy pairs. For example, a support material, also known as NV diamond (nitrogen vacancy diamond), can be excited with laser radiation with wavelengths in the visible range and also emits in the visible wavelength range (see, e.g., Weil, T. 2017: Nanodiamonds with lattice defects as innovative materials for biomedical applications; Research Report 2017 - Max Planck Institute for Polymer Research).

[0016] In further embodiments of the reference sample according to the invention, the carrier material is doped at least in regions with silicon vacancy pairs (SiV centers), phosphorus, oxygen, sulfur, tin and / or manganese vacancy pairs.

[0017] The reference sample can have one or more structures. These are each formed from at least one substructure, which can be configured differently to enable different measurements or to be usable for different optical systems, in particular different microscopes or cameras.

[0018] The structures are introduced into the substrate material of the support structure using a lithographic process, for example. The fabrication of such structures is described, for example, by Michl et al. (Michl, J. et al.: A Diamond Nanoscale Reference for Super-Resolution Microscopy, unpubl., submitted for publication in a scientific journal).

[0019] The lateral substructures of a structuring can be pairs of line segments (line pairs) arranged side by side, with a respective spacing between the line segments. This type of structuring, depending on the selected spacing of the line segments, makes it possible to determine whether the line segments of the respective substructures can be optically resolved. Depending on the extent of the structuring, it is also possible to check whether resolution is present only over certain areas or over the entire field of view. For example, using an appropriately dimensioned structuring, i.e., one comprising a correspondingly high number of substructures, it can be checked whether resolution is present in a central area of ​​the field of view as well as in a more peripheral area.The extent of the area of ​​a defect-free image, for example without distortions and / or without losses in the achievable resolution, can be used to determine the usable image field size.

[0020] To effectively determine the limit of resolution, it is advantageous to have several structures on a reference sample whose substructures or line segments are spaced at different distances from each other. For example, there can be at least one structure within which the spacing of the line segments of the substructures changes gradually from pair to pair. For example, the spacing of the line segments can be selected from a range of 25 nm to 500 nm and can be, for example, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, and / or 500 nm.

[0021] Using modern microstructuring techniques, lateral resolutions and positioning accuracies of 5 nm can be achieved. For example, the variance of the spacing of the line segments of the respective pairs is less than 2% or less than 2 nm, whichever is greater. The width of the lines of the substructures is advantageously no more than 50 nm, preferably no more than 20 nm, and their length is, for example, 10 µm. The individual pairs of line segments are spaced from each other with a constant pitch of, for example, 10 µm.

[0022] This design is extremely advantageous for comparing measuring instruments or optical systems and determining resolution, since the structure size has a significant influence on the perceived or measured resolution if it is equal to or slightly smaller than the size to be measured. Deviations from the desired structure shape / size are also subject to this limitation.

[0023] The substructures, particularly in their design as pairs of line segments, can be arranged parallel to each other. They can be perpendicular to a (virtual) reference line or inclined at a specific angle, for example, 30°, 45°, or 90°. In other designs, the substructures can also be arranged side by side in lines or rows.

[0024] In another possible embodiment, the substructures are formed, for example, as line segments that are parallel to one another and arranged in one direction with increasing spacing. A further embodiment is, for example, a two-dimensional grid whose grid lines are parallel to one another and arranged in at least one direction with increasing spacing.

[0025] With reference samples designed in this way, the respective optical transfer function (OTF), the modulation transfer function (MTF), and the point spread function (PSF) can be analyzed in addition to or as an alternative to determining the image field size. The MTF describes the contrast of all spatial frequencies of a system, for example, the number of line segments per unit length (e.g., per millimeter). The PSF specifies the imaging function of an image point. If the PSF is subjected to a Fourier transformation, the OTF is obtained. The OTF contains, in addition to the MTF, information on the so-called PTF (phase transfer function), in the sense of the relationship OTF = MTF*e^(i*PTF). The relationship can alternatively be expressed as { S ( z )} = ( k z ) e iΦ (k z)< with { S ( z )} as an optical transfer function, ( k z ) as modulation transfer function and e iΦ ( k z)< as a phase transfer function (see e.g. Hecht, E. (1987): Optics; 508 ff; ISBN: 0-201-11611-1).

[0026] In practical terms, this means that the MTF can be measured directly using a substructure in the form of line segments. The OTF depends significantly on the PSF of the system. The PSF can be determined using different substructures. For example, small point-like substructures can be used, which are approximately one-tenth to one-fifth of the minimum resolvable structure size of an optical system.

[0027] Such measurements are not only relevant for high-resolution optical systems, but can also be used for optical systems with different resolutions. The advantage here is the possible comparability and excellent calibration of the systems, which is based in particular on the robustness of the reference sample against unwanted bleaching.

[0028] The advantage is that not only a portion of the images is evaluated, but the optical system as a whole can be measured in a recording state or in a configuration state for actual use in fluorescence imaging. In contrast to the prior art, the reference sample according to the invention eliminates the need for separate recordings of illumination and detection beam paths, allowing direct and easily verifiable verification of a system's resolution capability.

[0029] In further embodiments of the invention, the lateral substructures of a respective structuring can be configured as surfaces that can be excited to emit luminescence. For example, such surfaces can be arranged in a grid. The individual surfaces of the grid advantageously have predetermined sizes, shapes, and distances from one another. Using such a grid, for example, distortions that occur can be detected, which are represented, for example, by curved outlines of the grid, even though the grid on the reference sample has straight outlines and right angles. The grids have dimensions of 250x250 µm, 1x1 mm, or 6x6 mm, for example. The pitches between the surfaces are, for example, 2 µm, 10 µm, or 25 µm, respectively. If the surfaces are configured as circular areas ("points"), these have diameters of, for example, 100 nm, 250 nm, or 500 nm.If the surfaces are formed as rectangles, their edge lengths are, for example, 100 nm, 250 nm, or 500 nm. Other possible designs include crosses, lines, or similar shapes. The thickness of the substructures in the z-direction is advantageously equal to or less than 50 nm, preferably equal to or less than 25 nm.

[0030] In further embodiments, the reference sample according to the invention can have a machine-readable coding as a structure. Such coding can be two-dimensional or three-dimensional and can be implemented, for example, in the form of so-called QR codes, barcodes, or individually defined shape and / or color combinations.

[0031] In a possible embodiment of the reference sample, the structuring has partial structures of a first lateral extent, for example a first diameter, a first circumference or a first edge length, in a central region and partial structures of a second lateral extent, for example a second diameter, a second circumference or a second edge length, in a peripheral region.

[0032] In further embodiments of the invention, the planar substructures can be designed, for example, as parts of a so-called Siemens star. The planar substructures, designed as circular sectors, are arranged alternately with circular sectors that cannot be excited to emit luminescence. The number of beams of the Siemens star is, for example, n = 16 with a diameter of, for example, 20 µm. Such a design enables a qualitative measurement of the resolution and a comparison of different microscopes.

[0033] In order to determine the resolution of a particular microscope along its optical axis (z-direction), the reference sample is provided with an axial substructure according to the invention. In an advantageous embodiment, this comprises at least one pair of lines intersecting at a single point. The lines run obliquely to the z-direction in the support material. The exact position of the intersection point, as well as the positions and paths, in particular the lengths, angles, and thicknesses or widths of the lines in the support material, are known.

[0034] Such a structuring with axial substructures allows the axial resolution of the microscope to be determined. In addition, a deviation of the current alignment of the microscope's optical axis relative to the intersection point can be determined. To do this, the microscope or its corresponding optical elements are focused in the z-direction. The focus is shifted in the z-direction and the resulting emission radiation is detected. If the intersection point is located exactly on the optical axis, luminescence is only recorded once when the focus is directed towards the intersection point and luminescence is excited there. If the path along which the focus is shifted in the z-direction is located away from the intersection point, luminescence is excited whenever the focus is directed towards one of the lines.The relative position of the reference sample and the optical axis can be determined from the position of the origins of the respective luminescence (= current focus position), the thickness of the lines and the distance between the two origins.

[0035] In addition, such an axial substructure allows the axial resolution to be measured. For this purpose, the reference sample is moved relative to the optical axis, in particular along an XY plane and thus orthogonal to the optical axis running in the z-direction. Since the positions and courses of the lines are known, it is possible, for example, to start from the intersection point and search for the (border) position at which the optical axis intersects both lines and both lines can still be distinguished from one another due to the fluorescence radiation emitted by each. The thickness of the lines must also be taken into account. Based on the distance of the border position thus found from the intersection point and with knowledge of the positions and courses of the lines, the distance between the lines at the border position can be determined. This distance represents the achievable axial resolution of the microscope in question.

[0036] In a further embodiment of the reference sample according to the invention, intersecting lines are designed as lateral substructures. The lines run parallel to a surface of the carrier material, in particular in an XY plane of the reference sample. Here, too, the resolution, this time in the lateral direction, can be determined based on the determined boundary position. The resolution can be calculated, for example, based on the known positions of the intersection point and boundary position, as well as the course of the lines. It is also possible to use a pre-prepared look-up table (LUT) for the lateral or axial resolution.

[0037] In a further embodiment of the reference sample according to the invention, at least one line also runs obliquely in the carrier material, but the lines intersect at a point outside at least one of the lines' centers. It is also possible for the at least one oblique line not to intersect the at least one other line, but merely to approximate it. If the distances between the lines at the different positions of the structuring are known, they can be used to determine the resolution, as described above.

[0038] In further embodiments, a structuring comprises axial substructures in the form of surfaces or cuboids. Their edge lengths are, for example, 100 nm, 250 nm, or 500 nm. In an alternative embodiment, the structuring is implemented as a surface, for example, 5 x 5 mm in size, and is created approximately 25 nm, but advantageously 10 nm, below the surface of the carrier material. The variance of the depth of the surface relative to the surface is preferably 5 nm or less.

[0039] If spheres are formed as axial substructures, they can have diameters of, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 1000, or 1400 nm. The spheres can be arranged in grids (arrays). Such a grid is, for example, 10 x 10 µm in size.

[0040] If the spheres are arranged linearly or in a grid, for example, in a 4x4 grid, the spacing between the spheres is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 1000, and / or 1400 nm. For all embodiments of the spheres, surfaces, or cuboids, their dimensions within an array or row can vary in other embodiments. For example, the dimensions increase in one direction.

[0041] The design of the substructures in the shape of at least one sphere allows the measurement of a point spread function (PSF) of the optical imaging system. The PSF can be used to calibrate the optical imaging system. Depending on their design, planar and / or grid-arranged substructures allow not only the measurement of lateral and / or axial resolution but also the determination of other optical effects, such as vignetting ("shading"). For example, at the same illuminance, a detected intensity at the edge of the field of view is determined relative to the center of the field of view.

[0042] In another possible embodiment of the reference sample, pairs of vacancies are generated as a structure across the surface. Within this area, which can be excited to emit luminescence radiation, there are regions in the form of, for example, circular areas, triangles, rectangles, polygons, and / or other shapes, where no pairs of vacancies are generated. These non-excitable regions can be arranged in a grid. As a result, the test pattern is thus designed, at least over a region of the field of view, as a matrix that can be excited to emit luminescence radiation and contains non-excitable regions. When using a reference sample with such a test pattern, the non-excitable regions within the excitable matrix are detected, and the resolution is determined.

[0043] The reference sample according to the invention can be used to calibrate a high-resolution microscope. A correspondingly designed method comprises the step of providing the reference sample in the beam path of the microscope to be calibrated, wherein at least one partial structure of a selected structuring of the reference sample is brought into an object field (field of view; FoV) of the microscope. Image data of the at least one partial structure is acquired by the microscope and evaluated by an analysis unit. The evaluation results are stored in a retrievable form and are thus available for downstream data processing. The analysis unit is, for example, a computer.

[0044] The reference sample can be provided in this and other processes by a user inserting and positioning the reference sample. It is also possible to use artificial intelligence algorithms to select and / or correctly position the reference sample. In further embodiments, recognition of the reference sample and its correct positioning can be achieved by recognizing, capturing, and evaluating a machine-readable code. Based on the evaluation results, the necessary control commands can be generated and, for example, the drives of a sample stage can be controlled accordingly.

[0045] Depending on the calibration currently being performed, the reference sample can be recognized and checked to determine which of the test samples it contains is suitable for the current calibration. If a suitable test sample is available, it is positioned so that the calibration can then be carried out. The individual calibrations can be carried out automatically one after the other. During or after a calibration, the user can be given information on the current status and result of the respective calibration to allow for success monitoring and any necessary user intervention. At the end of the calibration, the user can be shown a report on the calibration process, including a success rating and other parameters.

[0046] The same applies if a further calibration is to be carried out subsequently, for example to calibrate other components of the optical system, in particular the microscope.

[0047] The reference sample can also be used in a method for referencing at least two microscopes to one another. Such a method comprises the step of providing the reference sample in the beam path of a first microscope, wherein at least one partial structure of a selected structuring of the reference sample is brought into an object field (field of view; FoV) of the first microscope. Image data of at least one partial structure is acquired and evaluated using the first microscope, and the first evaluation results are stored in a retrievable form. Optionally, the setting or operating parameters of the first microscope can also be stored. In order to be able to compare two or more microscopes with one another, it is advantageous if the recording conditions for the images are as comparable as possible.For example, settings for the illumination power, pixel resolution, and parameters of the lens used can be saved in order to adjust them to the corresponding operating parameters of the second microscope, or at least to approximate them.

[0048] The reference sample is placed in the beam path of a second microscope, with at least one, preferably the same, partial structure of the selected structuring of the reference sample being brought into an object field (field of view; FoV) of the second microscope. Using the second microscope, image data of at least one partial structure is acquired and evaluated, and the evaluation results are provided as second evaluation results. The first and second evaluation results are compared with each other.

[0049] The reference sample can be detected and evaluated according to a predefined routine, which is stored, for example, in a control unit and called up as needed. Alternatively, a process can be implemented using artificial intelligence and / or neural networks, for example, to adapt detected parameters to the captured image content and optimize image acquisition.

[0050] In a further embodiment of the method, the first and / or second evaluation results are compared with expected values ​​using a comparison unit.

[0051] Additionally or alternatively, the microscope in question can be classified based on the evaluation or comparison results. For example, it can be assigned to predefined groups.

[0052] Based on the measured values, it is possible to derive technical parameters of the optical system and use them, for example, in the control of motorized components.

[0053] Furthermore, it is possible to adjust signal curves based on the determined parameters of the components involved and the specific optical effects recorded in order to improve the quality of the acquired image data.

[0054] In addition to automated calibration and adjustment, the results of the acquisition of the reference sample and its evaluation can be used for automated acquisition of specific sample areas.

[0055] It is also possible to create calibration curves, for example of a lighting output, and save them in a look-up table or a function.

[0056] A particular advantage of the invention is the use of a non-bleaching sample. This makes it possible to make direct comparisons between systems without degradation effects or uncertainties due to different luminescence responses at the same illuminance, for example, as a result of bleaching. Therefore, no waiting times for the sample to recover after use are required. The repeatability of measurements and comparisons is significantly improved. Furthermore, long-term exposures and comparisons of the long-term stability of the systems are very well possible. Test samples according to the invention can be produced with sizes in the lateral direction from 20 nm to 2 µm with high reproducibility and low deviations. In addition, the absorption spectra of the reference samples according to the invention in the various embodiments and their broad emission spectra open up the possibility of use in many different optical systems.

[0057] The invention is explained below using exemplary embodiments and illustrations. In the following: Fig. 1 shows a schematic representation of an embodiment of a reference sample according to the invention with five different structures, each formed from a number of partial structures; Fig. 2 shows a schematic representation of an embodiment of a microscope with a further embodiment of a reference sample according to the invention; Figs. 3a to 3e show schematic representations of five test patterns with increasing distances between the partial structures; line pairs arranged in a row are shown, in Fig. 3a as perpendicular or vertical line pairs, in Fig. 3b as inclined line pairs and in Fig. 3c as horizontal line pairs; Fig. 3d with increasing distances and Fig. 3e as a grating; Fig. 4 a schematic representation of an embodiment of a reference sample with a test pattern in the form of a Siemens star; Fig. 5 a schematic representation of an embodiment of a reference sample with a test pattern in the form of a grid of regularly arranged surfaces as substructures; Fig. 6 a schematic representation of an embodiment of a reference sample with a test pattern in the form of an inner grid of regularly arranged surfaces as first substructures and an outer grid with second substructures; Fig. 7 a schematic representation of an embodiment of a reference sample with a test pattern in the form of a grid of surfaces that cannot be excited to emit in a matrix that can be excited to emit; Fig.8 shows a schematic representation of a first exemplary embodiment of an axial partial structure in the form of two lines intersecting in the z-direction, as well as a method for determining the axial resolution; Figs. 9a and 9b show a schematic representation of a second exemplary embodiment of an axial partial structure in the form of two lines approaching each other in a perspective view (. Fig. 9a ) and in a top view ( Fig. 9b ); Fig. 10a and 10b show a schematic representation of a third embodiment of an axial substructure in the form of an arrangement of spherical axial substructures in a perspective view ( Fig. 10a ) and in a top view ( Fig. 10b ); Fig. 11 is a flowchart of an embodiment of a method for calibrating a microscope using a reference sample according to the invention; and Fig. 12 is a flowchart of an embodiment of a method for comparing a first microscope with a second microscope using a reference sample according to the invention.

[0058] The Fig. 1 The schematically shown reference sample 1 has a carrier structure 2 made of NV diamond as the carrier material 3. Applied to a side surface of the carrier structure 2 are first to fourth structurings 5, 6, 7, and 8 (= test samples 5, 6, 7, and 8, respectively), which are formed from substructures 5.1 in the form of lines arranged in a row, substructures 6.1 in the form of square surfaces, substructures 7.1 in the form of concentric rings, and substructures 8.1 in the form of point-shaped surfaces. In addition, there is a substructure that is designed as a machine-readable coding 17 in the form of a QR code. The side surface with the structurings 5, 6, 7, and 8 extends in a plane that is parallel to an XY plane of a Cartesian coordinate system.

[0059] The substructures 5.1 of the first structuring 5 are pairs of lines whose respective distance di (i = 1, 2, ..., n) varies from each other (see Figur 3a bis 3c ). The same applies to the fourth substructures 8.1. Likewise, the radii of the substructures 7.1 of the third test pattern 7 increase from the inside to the outside with increasing step sizes.

[0060] In the Fig. 2 The use of a reference sample 1 according to the invention for calibrating and / or adjusting a particularly high-resolution microscope 9 is illustrated. The microscope 9 has an evaluation and control unit 10, by means of which an evaluation of acquired image data as well as the generation and transmission of control commands is possible. The evaluation and control unit 10 is configured accordingly for this purpose. The acquisition of image data, and optionally also an illumination of the reference sample 1, is carried out by means of an objective 11, whose optical axis oA is directed in the z-direction onto the reference sample 1 and the structures 5, 7 and 8. This embodiment of the reference sample 1 does not have a second structure 6. A further or second microscope 9 (not shown) can have the same elements as in Fig. 2 shown.

[0061] The reference sample 1 is arranged on a sample stage 12. This stage can optionally be moved in the direction of at least one axis x, y, and z by means of the drives 13. The movement of the sample stage 12 can be controlled by control commands from the evaluation and control unit 10. A comparison unit 15, which is also present, is designed, for example, as a computer and is configured to compare evaluation results from the evaluation and control unit 10 with one another and to provide comparison results. The comparison unit 15 can also be configured to receive evaluation results from another optical system, for example, another microscope, and to compare them with evaluation results from the first microscope 9 (symbolized by the blind-ended connection).

[0062] For calibration purposes, at least one of the structures 5, 7, and 8 is illuminated with suitable excitation radiation, stimulating the emission of light, for example, fluorescent radiation. The emitted light is captured by the microscope 9, and the image data of the respective structure 5, 7, and / or 8 is analyzed in the evaluation and control unit 10 configured for this purpose. If the corresponding captured structures 5, 7, and / or 8, whose dimensions are known, can be resolved with a permissible degree of certainty or to a predetermined degree, the microscope 9 is correctly calibrated.

[0063] If, however, the recorded structures 5, 7, or 8 cannot be sufficiently resolved, control commands can be generated that move the sample stage 12, change technical parameters of the microscope 9, and / or adjust a computational analysis of the image data. These measures are performed to calibrate the microscope 9.

[0064] Alternatively or additionally, an adjustment can also be performed by bringing the optical axis oA of the lens 11 into a predetermined relative position to at least one of the substructures 5.1, 7.1 (not all labeled for reasons of clarity) and / or 8.1. The point-shaped substructures 8.1 of the test pattern 8 are particularly suitable for adjustment.

[0065] In the Fig. 3a bis 3c Three modifications of the first test pattern 5 are shown, each of which is formed as a number of pairs of line segments (line pairs) as partial structures 5.1. The term "line segments" is chosen due to the short lengths of the individual partial structures 5.1, which are approximately 10 µm. In all three versions ( Fig. 3a bis 3c ) a distance di (i = 1, 2, ..., n) of the line segments of a line pair increases from left to right (only a few examples are given). In Fig. 3a the distance d1 between the line segments of the first line pair is smaller than the distance d3 between the third line pair. Fig. 3a The partial structures 5.1 shown are parallel and vertically arranged line pairs with respect to a row direction from left to right. While the distances d1 to d4 change gradually in the row direction, the first line segments of a line pair are spaced from each other by a constant pitch a of, for example, 10 µm (shown only for the first and second line pair). The same applies to the embodiments of the Fig. 3b und 3c shown substructures 5.1. The Fig. 3b The pairs of lines shown are also parallel to each other, but inclined against a vertical direction. Fig. 3c Pairs of lines are arranged horizontally in a row.

[0066] A possible design with increasing distances between adjacent line sections is shown in the Fig. 3d shown. Such a further design of test pattern 5 can be used, for example, to determine the MTF.

[0067] A two-dimensional variation of a test pattern 5 with varying distances is shown in Fig. 3e realized in the form of a grid.

[0068] A further embodiment of a test pattern 6 according to the invention is a so-called Siemens star ( Fig. 4 ). This is composed of a number of wedge-shaped circular sectors which can be alternately excited (shown hatched) or not excited (not hatched) to emit luminescent light. In the exemplary embodiment shown, a total of eight wedge surfaces are present. In further exemplary embodiments, for example, sixteen wedge surfaces are formed (not shown for better clarity). The outer diameter of the Siemens star is, for example, 20 µm. A test pattern 6 in the form of a Siemens star allows the determination of the resolving power in a known manner by searching for the maximum achievable resolution on the detection side of the circular sectors tapering towards the center of the test pattern 6. In addition, the test pattern 6 in the form of the Siemens star can be used to calibrate a microscope 9 and / or to compare at least two microscopes 9 (see, for example, Fig. 11 and 12 ).

[0069] As already mentioned Fig. 1 As described, the test pattern 6 can be a grid of similar substructures 6.1. In the embodiment according to Fig. 5 The substructures 6.1 are squares arranged in a regular grid with a constant pitch a. The test pattern 6 shown allows for a check for distortions in the image. Thus, the correct, undistorted images of the outlines of the substructures 6.1 can serve as evidence of distortion-free imaging. Furthermore, the brightness on the optical axis and in the field can be determined, and any vignetting can be detected and recorded.

[0070] In further embodiments of the test pattern 6, the substructures 6.1 can be designed as points or circles (not shown).

[0071] In a reference sample 1, substructures 6.1 of different sizes and / or shapes can also be present within a test sample 6 ( Fig. 6 ). In this case, a number of substructures 6.1 can be arranged in an inner grid, for example in the form of squares with a first edge length. The substructures 6.1 of the inner grid have a constant first pitch a1 along the rows and columns of the grid. The inner grid is surrounded by an outer grid, which is formed by substructures 6.1 with a second edge length and which are spaced from one another by a second pitch a2. The second edge length and the second pitch a2 are greater than the first edge length or the first pitch a1 of the substructures 6.1 of the inner grid.

[0072] In a further embodiment of a reference sample 1 according to the invention, the partial structures 6.1 can be designed as surfaces that cannot be excited to emit luminescence radiation, for example as circular surfaces, which are embedded in a surface that can be excited to emit luminescence radiation (matrix 16) ( Fig. 7 ).

[0073] In addition or alternatively to lateral substructures 5.1, 6.1, 7.1, 8.1 for determining the resolution in lateral direction using lateral substructures 5.1, 6.1, 7.1, 8.1 (see Fig. 1 bis 3 ) a reference sample 1 according to the invention has at least one axial partial structure 5.1ax. In one embodiment ( Fig. 8 ), the axial test pattern 5ax is formed from two lines intersecting in the direction of the z-axis (z-direction) at an intersection point S, which lines are formed to run obliquely in the carrier material 3.

[0074] Is the microscope 9 (see Fig. 2 ) is arranged relative to the reference sample 1 such that its optical axis oA is directed towards the intersection point S of the partial structures 5.1ax (scenario I), luminescence light is produced only at this intersection point S and an image I is obtained from a z-position that corresponds to the position of the intersection point S in the z-direction (symbolized by an oval BI). The intersection point S is found by shifting the focus of the microscope 9 or the objective 11 in the z-direction. The z-position of the image BI1 can be compared with an expected z-position of the intersection point S and used to calibrate the microscope or adjust it.

[0075] If, on the other hand, the reference sample 1 and the optical axis oA are in a laterally shifted relative position (scenario II), luminescence is produced when the focus hits the partial structure 5.1ax running closer to the surface of the support structure 2. This luminescence is acquired as the first image B II1 and saved together with the corresponding z-position. If the focus is subsequently shifted further along the optical axis oA, no luminescence light is recorded over a distance that corresponds to a gap between the partial structures 5.1ax along the course of the optical axis oA. If the focus position coincides with the z-position of the partial structure 5.1ax lying deeper in the support structure 2, luminescence light is produced again. A second image B II2 is acquired and saved with the corresponding z-position.Based on the stored z-positions of the images B II1 and B II2 and on the fact that a first image B II1 was already acquired after the focus had moved a distance that is less than the distance required to reach the expected z-position of the intersection point, the current relative position of reference sample 1 and optical axis oA can be deduced.

[0076] These axial substructures 5.1ax are used to determine the axial resolution of microscope 9. The ability to resolve the resulting gap between the structures is a measure of the system's ability to distinguish sizes of structures axially from one another.

[0077] In a modification of the invention, the axial partial structures 5.1ax of the test pattern 5ax are also shown as two lines running obliquely in the carrier material 3 ( Fig. 9a ), but they do not intersect. The partial structures 5.1ax run three-dimensionally, ie in two planes tilted to each other, in the carrier material 3. In the Fig. 9b , which shows a top view of the support structure 2 in the z-direction, it can be seen that the substructures 5.1ax approach each other but do not intersect.

[0078] For the purpose of adjustment and / or calibration, in principle, as in Fig. 8 Instead of finding an intersection point S, the distance between the substructures 5.1ax can be used as a measure of the resolution or for the alignment.

[0079] Axial substructures 14.1 of a further axial test sample 14 can also be spheres with different diameters, as shown schematically in Fig. 10b The substructures 14.1 can be formed in a common z-position ( Fig. 10a ). In further versions of the test pattern 14, the balls can also be arranged at different depths, i.e. at different z-positions.

[0080] Based on the Fig. 11 An embodiment of a method according to the invention for calibrating a high-resolution or super-resolution optical device, in particular a microscope 9, will be explained. The following steps can each comprise a plurality of individual measures, which for the purpose of better clarity are Fig. 11 (correspondingly also in Fig. 12 ) are grouped into functional and graphical blocks.

[0081] Initially, a reference sample 1 according to the invention is inserted into the optical system to be calibrated. This insertion can be performed by a user or automatically. It is also possible to use artificial intelligence algorithms to select and / or correctly position the reference sample 1. A program configured to perform the measurement task or calibration is started and, if necessary, the measurement task is specified (step K1). An initial decision (KE1) to be made determines whether a reference run is necessary. If the answer is negative (N), the parameters for image acquisition are automatically selected and / or determined. A first image is then captured and saved (step K2).

[0082] If the first decision (KE1) is affirmed (Y), a referencing run is carried out in step K3 and / or a current position of the sample table 12 is determined before continuing with step K2.

[0083] The subsequent second decision step KE2 determines whether the reference sample 1 to be imaged is visible in the acquired image. If this is not the case (N), the third decision step KE3 decides whether a manual search will be performed in step K3 or an automated search will be performed in step K4. In both search alternatives, reference marks can be used, which are provided by a user in step K3 or by the optical system in step K4.

[0084] If reference sample 1 is included in the acquired image, the process can proceed directly to step K5. In this step, the system moves to existing reference marks to establish the desired initial position of sample stage 12, microscope 9, and reference sample 1. This is followed by moving to the structure 5, 6, 7, or 8 to be measured and capturing an image of it. The acquired image data is evaluated with regard to, for example, image content, the determination of distances di, brightness, homogeneity, etc. Additionally or alternatively, acquired structures 5, 6, 7, or 8 can be used as reference marks.

[0085] The results obtained in this way are output or stored and made available (step R1).

[0086] If the results from step R1 are not to be compared with theoretical values ​​(N) (fourth decision step KE4), the process can be terminated. However, if such a comparison is to be performed (Y), step K6 is executed. The comparison results can optionally be used to classify the system (KE5). If no classification is to be performed (N), the comparison results are output or stored for retrieval (R2), and the process is terminated.

[0087] Alternatively (Y), in step K7, the system is classified based on the degree of agreement with the theoretical value or the achievement of the theoretical value.

[0088] The comparison results and the generated classification are output or stored for retrieval (R3), and the process is terminated. The process steps can be repeated for a second microscope 9.

[0089] An embodiment of a method for comparing two optical systems, in particular two microscopes 9, is described with reference to Fig. 12described. The reference sample 1 is placed in a first microscope 9, the relevant sample locations, in particular the desired test pattern 5, 6, 7 or 8, are searched for, and the image acquisition is prepared. For this purpose, the necessary image acquisition parameters must be known or stored (step V1). Such parameters include, for example, the temporal resolution, the pixel resolution, the light intensity, the detector gain, illumination times, scanning speed, and spectral distribution. Image acquisition and an evaluation of the acquired image data are also subsumed under step V1. The image acquisition can encompass all spatial dimensions (2D, 3D) as well as a time course. The evaluation is directed, for example, at a feature classified as relevant. Such a feature is, for example, a recorded intensity profile of the test pattern 5, 6, 7 or 8, of which at least one distance between the maxima of the intensities is evaluated.

[0090] The evaluation can additionally or alternatively focus on the size of the regions of the substructures 5.1 to 8.1, 14.1 excited to emit luminescent light (e.g., using the half-width); the size of a gap between excited regions (e.g., using the Raleigh, Dawes, or Sparrow criterion); the uniformity / homogeneity of excited regions; and the temporal constancy or variation of the excited regions. The evaluation can be based on criteria such as the Nyquist-Shannon theorem, the signal-to-noise ratio, contrast, and the required resolution. Reference sample 1 is taken from the first microscope 9.

[0091] The determined parameters are output and stored (R1). In a first decision step VE1 of the method, the question must be answered as to whether a comparison of the evaluation results of the first microscope 9 with those of a second microscope 9 should be performed. If the answer is negative (N), a comparison with theoretical values ​​is performed in step V4. These can, for example, be the theoretically achievable resolution according to the Abbe criterion. The result of the comparison is output or a classification of the first microscope 9 is performed (R5), and the method is terminated.

[0092] If, however, the comparison with a second microscope 9 is affirmed (Y) in decision step VE1, the reference sample 1 is inserted into the second microscope 9, and in step V2, the measures corresponding to V1 are carried out. The results are output and stored (R2).

[0093] The system parameters of the first microscope 9 are applied, as far as possible, to the second microscope 9. For example, a temporal resolution, a pixel resolution, the light intensity, a detector gain, exposure times, scanning speed, and / or spectral distribution can be adopted. If necessary, the parameters used for image acquisition are adapted to the requirements of the second microscope 9. For example, an adaptation may be necessary when comparing a laser scanning microscope with a wide-field microscope or a high-resolution SR microscope with a laser scanning microscope or wide-field microscope.

[0094] Subsequently, the results of the first microscope 9 from R1 are compared with the results of the second microscope 9 from R2 in a step V3. The results from V3 are output as comparison values ​​between the two microscopes 9 (R4). In the subsequent step V4, the comparison with theoretical values ​​takes place, the results are output in R5, and the process is terminated. Reference symbol

[0095] 1Reference sample 2Support structure 3Support material 4Slide 5First structuring / first test pattern 5.1Partial structure (of the first structuring 5) 5Axaxial test pattern 5.1Axaxial partial structure 6Second structuring / second test pattern 6.1Partial structure (of the second structuring 6) 7Third structuring / third test pattern 7.1Partial structure (of the third structuring 7) 8Fourth structuring 8.1Partial structure (of the fourth structuring 8) 9Microscope 10Evaluation and control unit 11Objective 12Specimen stage 13Drives 14Fifth structuring / fifth test pattern 15Comparison unit 16Matrix 17Machine-readable coding oAOptical axis BIImage from scenario IB II1 First image from scenario IIB II2 Second image from scenario IIK1, V1Process steps; with i = 1 to 7 KEi, VEiDecision steps with i = 1 to 5 RiResult steps with i = 1 to 3 SIntersection

Claims

1. Reference sample (1) for calibrating and / or adjusting a microscope (9), - having at least one carrier structure (2) made of a carrier material (3) which, at least in regions of its extent, is excitable to emit luminescence light, wherein the carrier material (3) has at least one three-dimensional structure (5, 6, 7, 8, 14, 17) which is predetermined in terms of its dimensions and shape and consists of a number of substructures (5.1, 6.1, 7.1, 8.1, 14.1), characterized in that - the carrier material (3) is diamond or silicon carbide; - the carrier material (3) is doped in or around the regions of the three-dimensional structure (5, 6, 7, 8, 14, 17) in order to be excitable to emit the luminescence light; and - the respective substructure (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1) is formed laterally and / or axially as an area, as points, as a three-dimensional structure or as a combination of at least two line sections, wherein a lateral substructure (5.1, 6.1, 7.1, 8.1) extends on or under a surface of the reference sample and substantially parallel thereto, wherein the substructures (5.1, 6.1, 7.1, 8.1, 14.1) comprise axial substructures (5.1ax) which extend in or obliquely to a z-direction into the carrier material and which comprise at least one pair of intersecting lines.

2. Reference sample (1) according to Claim 1, characterized in that the carrier material (3) has regions in which it is doped with nitrogen-vacancy, silicon-vacancy, phosphorus-vacancy, oxygen-vacancy, sulphur-vacancy, tin-vacancy and / or manganese-vacancy pairs.

3. Reference sample (1) according to either of Claims 1 and 2, characterized in that the lateral substructures (5.1, 6.1, 7.1, 8.1) of a structure (5, 6, 7, 8) are pairs of line sections with a respective distance (di) between the line sections, wherein the distance (di) between the line sections changes step by step from pair to pair.

4. Reference sample (1) according to Claim 3, characterized in that the distances (di) are selected from a range from 25 nm to 500 nm.

5. Reference sample (1) according to either of Claims 1 and 2, characterized in that the lateral substructures (6.1) of a respective structure (6) are formed as areas which are excitable to emit luminescence.

6. Reference sample (1) according to Claim 5, characterized by a thickness of the substructures (6.1) in the z-direction of less than or equal to 50 nm, preferably less than or equal to 25 nm.

7. Reference sample (1) according to either of Claims 5 and 6, characterized in that the two-dimensional substructures (6.1) are arranged in alternation with areas which are not excitable to emit luminescence.

8. Reference sample (1) according to any of Claims 1 to 4, characterized in that the substructures (6.1) are formed as areas which are not excitable to emit luminescence and are embedded in a matrix (16) which is excitable to emit luminescence.

9. Reference sample (1) according to Claim 1, characterized in that a structure (17) is formed as a machine-readable code.

10. Method for calibrating a microscope (9), comprising the steps of: - providing the reference sample (1) according to any of Claims 1 to 9 in the beam path of the microscope (9) to be calibrated, wherein at least one substructure (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1) of a selected structure (5, 6, 7, 8, 14) of the reference sample (1) is brought into an object field of the microscope (9); - acquiring image data of the at least one substructure (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1) by means of the microscope (9); - evaluating the acquired image data; and - storing the evaluation results in a retrievable form.

11. Method according to Claim 10, wherein storing the evaluation results in a retrievable form is storing the evaluation results as first evaluation results and the method comprises the further following steps: - providing the reference sample (1) in the beam path of at least a second microscope (9), wherein at least one substructure (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1) of the selected structure (5, 6, 7, 8, 14) of the reference sample (1) is brought into an object field of the second microscope (9); - acquiring image data of at least one substructure (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1) by means of the second microscope (9); - evaluating the acquired image data and providing the evaluation results as second evaluation results; and - comparing at least the first and second evaluation results with each other.

12. Method according to Claim 11, characterized in that the first and / or the second evaluation results are compared with expected values by means of a comparison unit (15).