Reference sample for microscopy, method and use

Abstract

The invention relates to a reference sample (1) for calibrating and / or adjusting a microscope (9) and to the use of a reference sample (1). The latter has at least one carrier structure (2) made of a carrier material (3) which can be excited at least in its extension to emit luminescence and at least one two-dimensional and / or three-dimensional structure (5, 6, 7, 8, 14) composed of a plurality of substructures (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1). According to the invention, the carrier material (3) is diamond or silicon carbide and is doped in the region of or around the two-dimensional and / or three-dimensional structure (5, 6, 7, 8, 14) so as to be excitable to emit luminescence.

Description

[0001] The present invention relates to a reference sample and to the use of a reference sample.

[0002] Optical imaging systems, in particular high-resolution microscopes, can be calibrated and / or adjusted using reference samples. Such reference samples are equipped with two-dimensional structures in the form of test patterns, which are produced in or applied to a carrier material of the reference sample. These test patterns have substructures, the positioning of which on the reference sample, the dimensions and the distances between one another, in particular their lateral distances, are known in advance.

[0003] Reference samples in the form of cover slips or cuvettes, which are commonly used in the field of optical microscopy, for example, are known. They consist of glass and have two-dimensional test patterns (structures) that can be excited to emit fluorescent radiation. The resolution of the use configuration of the microscope, for example, can be estimated on the basis of the fluorescent radiation of the captured test patterns.

[0004] The excitation and emission of fluorescent radiation is based on doped glasses, in particular doped with known rare earths. Although the fluorescent centers generated in this way currently do not degrade as quickly under excitation radiation and environmental conditions as fluorescent markers based on protein or inorganic marker molecules, even doped glasses show that the emission intensity sometimes decreases significantly under multiple excitations (for example, Royon, A. and Converset, N. in 2017: Quality Control of Fluorescence Imaging Systems - A new tool for performance assessment and monitoring; Optik & Photonik 2 / 2017: 22-25).

[0005] To date, the structures used as test patterns have been introduced into the carrier material using technical methods, the resolution of which corresponds to the resolution of the optical system, in particular the microscope, to be measured, calibrated or adjusted. Therefore, metrological determination of the resolution of a high-resolution microscope with reference samples known from the prior art is only possible within a very limited range.

[0006] The present invention is therefore also based on the object of proposing a reference sample that enables an improved resolution measurement compared to the prior art. Furthermore, the present invention aims to create a possibility for comparative measurements on optical systems and imaging systems, in particular microscopes, which are not influenced by the degradation of the luminescence intensity from the sample.

[0007] This object is achieved by the subject matter described below. Advantageous refinements of the invention are found in the following description.

[0008] The reference sample is used for measuring, calibrating and / or adjusting an optical imaging system, such as a microscope, in particular a high-resolution microscope, and has at least one carrier structure made of a carrier material which is excitable at least in its range, i.e. at least in an area or section, to emit light (luminescence). The carrier material also has at least one two-dimensional and / or three-dimensional structure which is predetermined in its dimensions and shape and is composed of a plurality of substructures. This two-dimensional and / or three-dimensional structure forms a test pattern. The reference sample can have several structures which can also differ from one another in their dimensions and shape. These structures are also referred to below as test patterns.

[0009] According to the application, the carrier material consists of diamond, which can in particular be produced by technology. Other embodiments according to the application use silicon carbide (SiC) as a carrier material. In order to make the carrier material excitable to emit light, the carrier material is doped in the area of the structure or around it.

[0010] The carrier structure allows the test pattern to be repeatedly provided and used and contains or carries this structure. For example, the carrier structure can be placed on a slide which is commonly used in microscopy.

[0011] The respective substructure is formed laterally and / or axially as an area, as a point, as a three-dimensional structure and / or as a combination of at least two line sections. In this case, the lateral substructure extends on or below the surface of the reference sample and substantially parallel thereto, while the axial substructure extends into the carrier material in the z direction or at an angle thereto.

[0012] An optical imaging system, for example a microscope, is referred to as high-resolution if it can be used to display structures smaller than the Abbe limit. The application will be explained below using the example of one or more microscopes.

[0013] The excitation to emit luminescence takes place in particular by means of excitation radiation with a suitable wavelength and intensity.

[0014] In an advantageous embodiment of the reference sample, the carrier material is doped with nitrogen vacancy pairs. For example, the carrier material, also known as NV diamond (nitrogen-vacancy diamond), is excitable with laser radiation having a wavelength in the visible range and likewise emits in the visible wavelength range (see, for example, Weil, T. in 2017: Nanodiamanten mit Gitterdefekten als innovative Materialien für biomedizinische Anwendungen; Research Report 2017 - Max-Planck-Institut für Polymerforschung).

[0015] In other embodiments of the reference sample according to the application, the carrier material is doped at least in regions having silicon vacancy pairs (SiV centers), phosphorus vacancies, oxygen vacancies, sulfur vacancies, tin vacancies and / or manganese vacancy pairs.

[0016] The reference sample can have one or more structures. These are formed by at least one substructure, which can be designed differently in order to carry out different measurements or can be used for different optical systems, in particular different microscopes or cameras.

[0017] The structures are introduced into the carrier material of the carrier structure, for example by means of a lithographic method. For example, Michl et al. (Michl, J. et al.: A Diamond Nanoscale Reference for Super-Resolution Microscopy, unpublished, submitted to a journal for publication) describe the manufacture of such structures.

[0018] The lateral substructures of the structure can be pairs of line segments (line pairs) arranged next to one another, the pairs of line segments having a distance between the respective line segments. This embodiment of the structure makes it possible to determine, depending on the distance between the selected line segments, whether the line segments of the respective substructure can be optically resolved. Depending on the extent of the structure, it can additionally be examined whether the resolution is present only over a region or over the entire field of view. For example, it can be examined by means of a structure of appropriate size, that is to say a structure comprising a corresponding number of substructures, whether the resolution is present, for example, in a central region of the field of view and in more peripheral regions. The extent of the region in which error-free imaging is possible, for example without distortion and / or without loss in the achievable resolution, can be used to determine the image region size that can be used in each case.

[0019] In order to find the resolution limit effectively, it is advantageous if there are a plurality of structures on the reference sample, the substructures or line segments of which are located at different distances from one another. For example, there can be at least one structure in which the distance between the line segments of the substructures changes stepwise with different pairs. For example, the distance between the line segments can be selected from the range from 25 nm to 500 nm and can be, for example, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400 and / or 500 nm.

[0020] Using modern microstructuring techniques, a lateral resolution and positioning accuracy of 5 nm can be achieved. In this case, for example, the distance between a respective pair of line sections varies by less than 2% or less than 2 nm, depending on which value is greater. The width of the lines of the substructure is advantageously no greater than 50 nm, preferably no greater than 20 nm, and their length is, for example, 10 pm. Individual pairs of line sections are spaced apart from one another at a constant pitch of, for example, 10 pm.

[0021] This embodiment has great advantages for the measurement of the resolution and the comparison of the optical system, since if the structure size is the same as or slightly smaller than the size to be determined, this structure size has a significant influence on the perceived or determined resolution. Deviations from the required structure shape / size are also subject to this restriction.

[0022] The substructures, in particular when they are embodied as pairs of line sections, can be arranged parallel to one another. They can be perpendicular to the (virtual) reference line or inclined at a specified angle, for example 30°, 45° or 90°. In other embodiments, the substructures can also be arranged adjacent to one another in the form of lines or rows.

[0023] In other possible embodiments, the substructures are formed, for example, as line sections which are arranged parallel to one another and arranged such that their distance from one another increases in one direction. Other embodiments are, for example, two-dimensional grids whose grid lines are parallel to one another and arranged such that their distance from one another increases in at least one direction.

[0024] Using a reference sample designed in this way, in addition to or as an alternative to the determination of the image field size, it is possible, for example, to analyze the corresponding optical transfer function (OTF), modulation transfer function (MTF) and point spread function (PSF). Here, the MTF describes the contrast of all spatial frequencies of the system, for example the number of line sections per unit length (for example per mm). The PSF specifies the mapping function of an image point. If the PSF is Fourier-transformed, the OTF is obtained. In addition to the MTF, the OTF also contains information about the known PTF (phase transfer function) in the sense of the connection OTF = MTF * e^(i*PTF). Alternatively, the connection can also be expressed as where as the optical transfer function, as the modulation transfer function, and as the phase transfer function (see, for example, Hecht, E. (1987): Optics; 508 ff; ISBN: 0-201-11611-1).

[0025] In practice, this means that the MTF can be measured directly by means of substructures in the form of line segments. The OTF is largely dependent on the PSF of the system. The PSF can be determined using different substructures. For example, small point-like substructures can be used for this, which for example correspond to approximately one-tenth to one- fifth of the smallest resolvable structure size of the optical system.

[0026] This measurement is 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 of the systems and the very good calibrability, which is based in particular on the robustness of the reference sample against unwanted quenching.

[0027] Advantageously, not only a part of the image is estimated, but the entire optical system can be measured in the recording state or configuration for the actual use of recording fluorescence images. In comparison to the prior art, using the reference sample according to the application, it is not necessary to record the illumination and detection beam paths separately and a direct and easily traceable proof of the resolution of the system can be provided.

[0028] In other embodiments of the application, the lateral substructures of the respective structure can be formed as regions which can be excited to emit luminescence. For example, these regions can be arranged in a grid. The individual regions of the grid advantageously have a predetermined size, shape and distance from one another. For example, even if the grid on the reference sample has straight contours and right angles, such a grid can be used to detect any occurring distortions, for example, in the case of curved contours of the grid. For example, the grid has dimensions of 250 x 250 pm, 1 x 1 mm or 6 x 6 mm. The pitch between the regions is for example 2 pm, 10 pm or 25 pm. If the regions are in the form of circular regions ("dots"), they have for example a diameter of 100 nm, 250 nm or 500 nm. If the regions are formed as rectangles, their edge length is for example 100 nm, 250 nm or 500 nm. Other possible embodiments are for example crosses, lines or similar shapes. The thickness of the substructures in the z-direction is advantageously less than or equal to 50 nm, preferably less than or equal to 25 nm.

[0029] In other embodiments, the reference sample according to the application can have a machine-readable code as a structure. Such a code can be a two- or three-dimensional code and can be in the form of for example known QR codes, barcodes or individually defined shape and / or color combinations.

[0030] In one possible embodiment of the reference sample, the structure has substructures of a first lateral extent (for example a first diameter, a first circumference or a first edge length) in a central region and substructures of a second lateral extent (for example a second diameter, a second circumference or a second edge length) in a peripheral region.

[0031] In other embodiments of the application, the two-dimensional substructures can for example be formed as parts of a known Siemens star. The two-dimensional substructures designed as sectors are arranged alternately with sectors that cannot be excited to emit luminescence. The Siemens star has for example a number of rays n = 16, and a diameter of for example 20 pm. Such embodiments enable a qualitative measure of the resolution and a comparison of different microscopes.

[0032] In other embodiments according to the application, the reference sample is equipped with an axial substructure in order to be able to determine the resolution of the respective microscope in the direction of its optical axis (z direction). In an advantageous embodiment, this comprises at least one pair of lines that intersect at one point. The lines extend in the carrier material inclined to the z direction. The exact position of the intersection point as well as the position and distribution, in particular the length, angle and thickness or width of the lines in the carrier material, are known.

[0033] This embodiment of the structure with an axial substructure allows the axial resolution of the microscope to be determined. In addition, the deviation of the instantaneous alignment of the optical axis of the microscope or its corresponding optical elements with respect to the intersection point can be determined. For this purpose, the microscope or its corresponding optical elements are focused in the z direction. The focus point is displaced in the z direction, and the resulting emitted radiation is detected. If the intersection point happens to lie on the optical axis, only one luminescence is captured when the focus point is directed at the intersection point and luminescence is excited there. If the path of the focus point displaced in the z direction is away from the intersection point, luminescence is excited when the focus point is directed at one of the lines. The relative positional relationship of the reference sample to the optical axis can be inferred from the position of the respective luminescence source (= current focus point position), the thickness of the lines and the distance between the two origins.

[0034] In addition, this axial substructure allows the axial resolution of the measurement. For this purpose, the reference sample is displaced with respect to the optical axis, in particular along the XY plane, and thus orthogonally to the optical axis extending in the z direction. Since the position and distribution of the lines are known, it is possible, for example, to search for the (boundary) position at which the optical axis intersects two lines starting from the intersection point, and the two lines can still be distinguished from each other on the basis of the fluorescent radiation emitted by them. The thickness of the lines must also be taken into account. On the basis of the distance of the boundary position found in this way from the intersection point, and in the knowledge of the position and distribution of the lines, the distance between the lines at the boundary position can be determined. This distance reflects the axial resolution that can be achieved by the microscope in question.

[0035] In other embodiments of the reference sample according to the application, the intersecting lines are implemented as a lateral substructure. The lines run parallel to the surface of the carrier material, in particular in the XY plane of the reference sample. Here too, the resolution can be determined on the basis of the limit positions determined, this time in the lateral direction. For example, the known position of the intersection point and the limit positions as well as the distribution of the lines can be used to calculate the resolution. A previously compiled look-up table (LUT) can also be used for the lateral or axial resolution.

[0036] In other embodiments of the reference sample according to the application, at least one line likewise extends obliquely in the carrier material, but these lines intersect at a point other than at least one of the midpoints of the lines. It is likewise possible for at least one oblique line to not intersect at least one other line but only to approach it. If the distances between the lines at different positions of the structure are known, they can be used to determine the resolution, as described above.

[0037] In other embodiments, the structure comprises axial substructures in the form of regions or cuboids. For example, they have an edge length of 100 nm, 250 nm or 500 nm. In alternative embodiments, the structure is realized as a region which has dimensions of, for example, 5 x 5 mm and is produced approximately 25 nm, but advantageously 10 nm, below the surface of the carrier material. The variation in the depth of the region relative to the surface is preferably 5 nm or less.

[0038] If the spheres form axial substructures, for example the diameter of these substructures can be 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 a grid (array). The dimensions of such a grid are, for example, 10 x 10 pm.

[0039] If the spheres are arranged linearly or in a grid, for example in a 4 x 4 grid, the distance 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. In the case of all embodiments of spheres or regions or cuboids, the extent of these within the array or line can differ in other embodiments. For example, the extent increases in one direction.

[0040] Embodiments of at least one substructure in the form of a sphere allow the point spread function (PSF) of an optical imaging system to be measured. The PSF can be used to calibrate the optical imaging system. Depending on the configuration, two-dimensional and / or grid-like substructures allow not only the lateral and / or axial resolution to be measured, but also other optical effects, such as vignetting (“shading”) to be determined. In this case, for example, the detected intensity is determined at the periphery of the field of view relative to the center of the field of view in the case of the same illumination.

[0041] In other possible embodiments of the reference sample, pairs of voids are generated two-dimensionally as structures. Within the area that can be excited to emit luminescence radiation, there are, for example, circular areas, triangular, rectangular, polygonal and / or other shaped areas on which no pairs of voids are generated. These non-excitable areas can be arranged in a grid. The test pattern is thus designed at least over the area of the field of view to be excitable to emit luminescence radiation and in a matrix with which non-excitable areas are present. When using a reference sample with such a test pattern, non-excitable areas within the excitable matrix are detected and the resolution is determined.

[0042] The reference sample according to the application can be used for calibrating 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 substructure of a selected structure of the reference sample is introduced into the object field (field of view; FoV) of the microscope. Image data of the at least one substructure are acquired using the microscope and evaluated by means of an analysis unit. The evaluation result is stored in a retrievable form and thus usable for subsequent data-technical processing. The analysis unit is, for example, a computer.

[0043] The reference sample can be provided in this and other methods by the user placing and positioning the reference sample. Artificial intelligence algorithms can also be used to select and / or correctly position the reference sample. In other configurations, the reference sample can be identified and correctly positioned by detecting, capturing and evaluating machine-readable codes. Necessary control commands can be generated on the basis of the evaluation result and, for example, the drive of the sample stage can be controlled accordingly.

[0044] Depending on the calibration currently to be carried out, it can be identified and checked which test pattern contained in the reference sample is suitable for the current calibration. If a suitable test pattern is available, it is positioned so that the calibration can then be carried out. Individual calibrations can be carried out automatically one after the other. During or after the calibration, the user can be given information about the current status and the corresponding calibration result in order to allow successful monitoring and to make it possible for the user to carry out any necessary interventions. At the end of the calibration, the user can be shown a report of the calibration process with a success rating and other parameters.

[0045] The same applies if further calibrations are then to be carried out, for example in order to calibrate further components of the optical system, in particular further components of the microscope.

[0046] The reference sample can also be used in a method in which at least two microscopes are referenced to one another. This method comprises the step of providing the reference sample in the beam path of a first microscope, wherein at least one substructure of a selected structure of the reference sample is introduced into the object field (field of view; FoV) of the first microscope. By means of the first microscope, image data of the at least one substructure are acquired and evaluated, and the first evaluation result is stored in a retrievable form. Optionally, the settings 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 of the images are as comparable as possible. For example, the settings of the illumination power, the pixel resolution and the objective parameters used can be stored in order to set them or at least to approximate them with the corresponding operating parameters of the second microscope.

[0047] The reference sample is provided in the beam path of a second microscope, wherein at least one (preferably the same) substructure of a selected structure of the reference sample is again introduced into the object field (field of view; FoV) of the second microscope. Using the second microscope, image data of the at least one substructure are acquired and evaluated, and the evaluation result is provided as a second evaluation result. The first and second evaluation results are compared with one another.

[0048] The reference sample can be identified and evaluated according to a predefined routine, which is stored, for example, in the control unit and retrieved when required. Alternatively, a method using artificial intelligence and / or neural networks can be implemented in order to, for example, adapt the captured parameters to the recorded image content and optimize the image capture.

[0049] In other configurations of the method, the first and / or second evaluation result is compared with an expected value by means of a comparison unit.

[0050] Additionally or alternatively, the microscopes in question can be classified on the basis of the evaluation result or the comparison result. To this end, for example, the microscopes can be assigned to predefined groups.

[0051] On the basis of the determined measurement values, technical parameters of the optical system can be derived and used, for example, to control electric components or the like.

[0052] Furthermore, signal curves can be adjusted on the basis of the determined parameters of the components involved and the detected specified optical effects in order to improve the quality of the acquired image data.

[0053] In addition to the automatic calibration and adjustment, the capture results of the reference sample and their evaluation can also be used for the automatic capture of specified sample regions.

[0054] Furthermore, calibration curves, for example, of the illumination power, can be created and stored in, for example, look-up tables or functions.

[0055] The advantages of the present application are in particular the use of quenching-free samples. This makes it possible to make direct comparisons between systems without degradation effects or uncertainties due to different luminescence responses at the same illumination (for example due to quenching). Thus, there is no need to observe a waiting time for the samples to recover after they have been used. The repeatability of the measurements and comparisons is significantly improved. Furthermore, comparisons of the very good long-term exposure and long-term stability of the systems are possible. The test patterns according to the present application can be manufactured with high repeatability and small deviations in the size in the lateral direction of 20 nm to 2 pm. Furthermore, the absorption spectra of the reference samples according to the present application and their broad emission spectra in different embodiments make the use in many different optical systems possible.

[0056] The application is explained in more detail below on the basis of exemplary embodiments and the accompanying drawings, in which:

[0057] Figure 1 A schematic diagram of an exemplary embodiment of a reference sample according to the present application is shown, which has five different structures, each of which is composed of a plurality of substructures;

[0058] Figure 2 A schematic diagram of an exemplary embodiment of a microscope with other exemplary embodiments of reference samples according to the present application is shown;

[0059] Figures 3a to 3e Five test patterns with increasing distances between the substructures are shown; line pairs arranged in a row are shown, Figure 3a vertical or upright line pairs in, Figure 3b inclined line pairs in, Figure 3c horizontal line pairs in ; Figure 3d the distances increase in, and Figure 3e a grid in ;

[0060] Figure 4 A schematic diagram of an exemplary embodiment of a reference sample with a test pattern of Siemens stars is shown;

[0061] Figure 5 A schematic diagram of an exemplary embodiment of a reference sample with a test pattern in the form of a grid of regularly arranged areas as substructures is shown;

[0062] Figure 6 A schematic diagram of an exemplary embodiment of a reference sample with a test pattern in the form of an inner grid of regularly arranged areas as first substructures and an outer grid with second substructures is shown;

[0063] Figure 7 A schematic diagram of an exemplary embodiment of a reference sample with a test pattern in the form of a grid of areas that cannot be excited to emit as substructures in a matrix that can be excited to emit is shown;

[0064] Figure 8 A schematic diagram of a first exemplary embodiment of an axial substructure in the form of two lines intersecting in the z-direction, and a procedure for determining the axial resolution;

[0065] Figure 9a and 9b A schematic diagram of a second exemplary embodiment of an axial substructure in the form of two lines approaching each other is shown in perspective view Figure 9a ) and plan view Figure 9b ) ;

[0066] Figure 10a and 10b A schematic diagram of a third exemplary embodiment of an axial substructure in the form of an arrangement of spherical axial substructures is shown in perspective view Figure 10a ) and plan view Figure 10b ) ;

[0067] Figure 11 A flowchart of a version of the method of calibrating a microscope with reference to a sample according to the application is shown; and

[0068] Figure 12 A flowchart of a version of the method of comparing a first microscope with a second microscope using a reference sample according to the application is shown.

[0069] Figure 1 The reference sample 1 is shown schematically and has a carrier structure 2 made of NV diamond as a carrier material 3. Applied to the side surface of the carrier structure 2 are first to fourth structures 5, 6, 7 and 8 (respectively = test patterns 5, 6, 7 and 8) which consist of substructures 5.1 in the form of lines arranged in rows, substructures 6.1 in the form of rectangular areas, substructures 7.1 in the form of concentric rings, and substructures 8.1 in the form of dot-shaped areas. In addition, there are substructures in the form of a machine-readable code 17 in the form of a QR code. The side surface with the structures 5, 6, 7 and 8 extends in a plane parallel to the XY plane of a Cartesian coordinate system.

[0070] The substructures 5.1 of the first structure 5 are line pairs whose respective distances di (i = 1, 2,..., n) to each other vary (see Figures 3a to 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 out with increasing increments.

[0071] Figure 2The use of the reference sample 1 according to the application for calibrating and / or adjusting, in particular, a high-resolution microscope 9 is shown. The microscope 9 has an evaluation and control unit 10, by means of which an evaluation of the acquired image data and the generation and transmission of control commands are possible. The evaluation and control unit 10 is correspondingly configured for this purpose. The acquisition of the image data and optionally the illumination of the reference sample 1 takes place by means of an objective 11, the optical axis oA of which points in the z direction to the reference sample 1 and the structures 5, 7 and 8. This exemplary embodiment of the reference sample 1 does not have the second structure 6. A further or second microscope 9 (not shown) can have the same elements as shown in Fig. 1. Figure 2

[0072] The reference sample 1 is arranged on a sample stage 12. The latter is optionally movable in a controlled manner in the direction of at least one axis x, y and z by means of a drive 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 is likewise present, for example in the form of a computer and configured for comparing the evaluation results of the evaluation and control unit 10 with one another and making the comparison results available. The comparison unit 15 can additionally be implemented to receive evaluation results from another optical system, for example another microscope, and to compare them with the evaluation results from the first microscope 9 (represented by the dead-end connection).

[0073] For the purpose of calibration, at least one of the structures 5, 7 and 8 is illuminated with suitable excitation radiation and the emission of excitation light, for example fluorescent radiation. The emitted light is captured with the microscope 9 and the image data of the relevant structure 5, 7 and / or 8 are analysed in the evaluation and control unit 10 configured for this purpose. If the correspondingly captured structures 5, 7 and 8 of known size can be resolved with the permitted certainty or predetermined degree, the microscope 9 is correctly calibrated.

[0074] On the other hand, if the captured structures 5, 7 and 8 cannot be sufficiently resolved, control commands can be generated which result in the movement of the sample stage 12, a change in the technical parameters of the microscope 9 and / or the calculated evaluation of the image data is adjusted. These measures are taken in order to calibrate the microscope 9.

[0075] Alternatively or additionally, an adjustment can also be made by introducing the optical axis oA of the objective 11 into a predetermined position relative to at least one of the substructures 5.1, 7.1 (not all of which are marked for the sake of clarity) and / or 8.1. The punctiform substructures 8.1 of the test pattern 8 are particularly suitable for the adjustment.

[0076] Figures 3a to 3c ​Three modifications of the first test pattern 5 are shown, each of which is formed as a plurality of pairs (line pairs) of line segments of the substructure 5.1. The term line segment is chosen because the individual substructure 5.1 is short in length, approximately 10 pm. In all three embodiments Figures 3a to 3c ), the distance di (i = 1, 2,..., n) between the line segments of the line pairs increases from left to right (a small number is specified by way of example only). In Figure 3a , the distance d1 between the line segments of the first line pair is smaller than the distance d3 of the third line pair. Figure 3a The substructures 5.1 shown in Figure 3b and 3c are line pairs that are parallel to one another and arranged vertically with respect to the row direction from left to right. While the distances d1 to d4 change stepwise in the row direction, the respective first line segments of the line pairs are spaced apart from one another at a constant pitch a, which is, for example, 10 pm (shown only for the first and second line pairs). This applies equally to the embodiments of the substructure 5.1 shown in Figure 3b . The line pairs shown in Figure 3c are likewise parallel to one another, but are inclined with respect to the vertical direction. In , the line pairs are arranged in rows horizontally.

[0077] Figure 3d A possible embodiment of increasing distances between adjacent line segments is shown in .

[0078] A two-dimensional modification of the test pattern 5 with varying distances is implemented in the form of a grid in Figure 3e .

[0079] Further exemplary embodiments of the test pattern 6 according to the application are so-called Siemens stars Figure 4 . The latter consist of a number of wedge-shaped sectors, which are alternately excitable (shown in hatching) and non-excitable (unhatched) to emit luminescence. In the exemplary embodiment shown, there are a total of eight wedge-shaped regions. In other exemplary embodiments, for example, sixteen wedge-shaped surfaces are formed (not shown for a better overview). The outer diameter of the Siemens star can be, for example, 20 pm. The test pattern 6 in the form of a Siemens star allows the resolution to be determined in a known manner by searching for the maximum achievable resolution of the sectors that narrow towards the center of the test pattern 6 on the detection side. In addition, the test pattern 6 in the form of a Siemens star can be used to calibrate the microscope 9 and / or to compare at least two microscopes 9 (see, for example, Figure 11 and 12 ).

[0080] As already described with regard to Figure 1 , the test pattern 6 can be a grid of the same type of substructure 6.1. In accordance with Figure 5In exemplary embodiments of the reference sample 1, the substructures 6.1 are squares, which are arranged in a regular grid with a constant pitch a. The test pattern 6 shown allows to check for the occurrence of distortions in the image. Thus, a correct, undistorted image of the profile of the substructures 6.1 can serve as evidence of an undistorted image. Furthermore, the luminance on the optical axis and in the field can be determined, and any vignetting can be detected and captured.

[0081] In other embodiments of the test pattern 6, the substructures 6.1 can be in the form of dots or circles (not shown).

[0082] In the reference sample 1, substructures 6.1 of different sizes and / or different shapes can also be present within the test pattern 6 Figure 6 ). A plurality of substructures 6.1 can here 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 having a second edge length and being spaced apart from one another by a second spacing a2. The second edge length and the second pitch a2 are greater than the first edge length and the first pitch a1 of the substructures 6.1 of the inner grid.

[0083] In other embodiments of the reference sample 1 according to the application, the substructures 6.1 can be formed as regions that are not excitable to emit cold light radiation, for example in the form of circular regions embedded in regions excitable to emit cold light radiation (matrix 16) Figure 7 ).

[0084] In addition to determining the resolution in the lateral direction by means of the lateral substructures 5.1, 6.1, 7.1, 8.1 (see Figure 1 to 3), the reference sample 1 according to the application can additionally or alternatively have at least one axial substructure 5.1 ax. In exemplary embodiments Figure 8 ), the axial test pattern 5ax is formed by two lines that intersect in the z-axis direction (z direction) at a crossing point S and are formed to extend obliquely in the carrier material 3.

[0085] If the microscope 9 (see Figure 2 ) is arranged relative to the reference sample 1 such that its optical axis oA points to the crossing point S of the substructures 5.1 ax (scene I), cold light is generated only at this crossing point S and an image I (denoted with the oval BI) is obtained from a z position corresponding to the position of the crossing point S in the z direction. The crossing point S is here found by displacing the focal point of the microscope 9 or the objective 11 in the z direction. The z position of the image BI1 can be compared with the expected z position of the crossing point S and used for calibrating or adjusting the microscope.

[0086] If, on the other hand, the reference sample 1 and the optical axis oA are in a laterally displaced relative position (scenario II), luminescence occurs when the focal point is incident on a substructure 5.1 ax which extends close to the surface of the carrier structure 2. This luminescence is shown as being captured as a first image B Ⅱ1 and the associated z-position. If the focal point is then displaced further along the optical axis oA, no luminescence is captured along the path of the optical axis oA, along the path distance corresponding to the intermediate space of the substructure 5.1 ax. If the focal point position coincides with the z-position of a substructure 5.1 ax which is located deeper in the carrier structure 2, luminescence occurs again. A second image B Ⅱ2 is captured and the associated z-position is stored. The current relative position of the reference sample 1 and the optical axis oA can be inferred on the basis of the stored z-positions of the images B II1 and B II2 and on the basis of the fact that the second image B II1 was captured after the focal point has travelled a displacement distance which is less than the displacement distance required to reach the expected z-position of the intersection point.

[0087] These axial substructures 5.1 ax serve to determine the axial resolution of the microscope 9. The ability to resolve the gap which arises between the structures is a measure of the axial discrimination structure size of the system.

[0088] In a modification of the invention, the axial substructures 5.1 ax of the test pattern 5ax are likewise implemented as two lines which extend obliquely in the carrier material 3 Figure 9a but they do not intersect. The substructures 5.1 ax extend three-dimensionally, i.e. in two mutually oblique planes, in the carrier material 3. Figure 9b which shows a plan view of the carrier structure 2 in the z-direction, shows that the substructures 5.1 ax approach one another but do not intersect.

[0089] In principle, the procedure described for Figure 8 can be used for adjustment and / or calibration purposes. Instead of finding the intersection point S, the distance between the substructures 5.1 ax can be used as a measure of the resolution or adjustment.

[0090] The axial substructures 14.1 of the further axial test pattern 14 can also be spheres with different diameters, as Figure 10b is shown schematically. The substructures 14.1 can be formed at a common z-position Figure 10a In other embodiments of the test pattern 14, the spheres can also be arranged at different depths, i.e. at different z-positions.

[0091] Reference will be made to Figure 11An explanation of a version of the method for calibrating a high-resolution or super-resolution optical device, in particular a microscope 9, according to the invention. Each of the subsequent steps can comprise a plurality of individual measures, which, for the sake of clarity, are combined functionally and graphically into blocks in Figure 11 Figure 12

[0092] At the beginning, a reference sample 1 according to the invention is placed into the optical system for calibration. The placement can be done by the user or automatically. Artificial intelligence algorithms can also be used to select and / or correctly position the reference sample 1. A program set up for carrying out a measurement task or a calibration is started and, if necessary, the measurement task is specified (step K1). In a first decision (KE1) to be made, it is determined that a reference run needs to be carried out. If the answer is no (N), the parameters for the image recording are automatically selected and / or determined. A first image is then captured and stored (step K2).

[0093] If the first decision (KE1) is yes (Y), the reference run is carried out in step K3 and / or the current position of the sample table 12 is determined before continuing with step K2.

[0094] The subject of the subsequent second decision step KE2 is the question of whether the reference sample 1 to be imaged is visible in the captured image. If this is not the case (N), a decision is made in a third decision step KE3 whether a manual search is carried out in step K3 or an automatic search is carried out in step K4. In both alternatives of the search, a reference mark can be used, which is provided by the user in step K3 or by the optical system in step K4.

[0095] If the reference sample 1 is contained in the captured image, the process can continue directly with step K5. In this step, the existing reference mark is homed in order to produce the desired starting position of the sample table 12, the microscope 9 and the reference sample 1. The structure 5, 6, 7 or 8 to be measured is homed and an image thereof is recorded. The acquired image data are evaluated, for example in terms of image content, determination of distances di, brightness, uniformity, etc. In addition or alternatively, the captured structure 5, 6, 7 or 8 can be used as a reference mark.

[0096] The results obtained in this way are output or stored in a retrievable manner and are available (step R1).

[0097] ​​If the result from step R1 is no (N) to be compared with the theoretical value (fourth decision step KE4), the method can end. If, on the other hand, such a comparison is to be carried out (Y), step K6 is carried out. The comparison result can optionally be used to classify the system (KE5). If no classification is carried out (N), the comparison result is output or stored in a retrievable manner (R2) and the method ends.

[0098] Alternatively (Y), in step K7, the system is classified based on the degree of agreement with the theoretical value or whether the theoretical value is reached.

[0099] The comparison result and the resulting classification are output or stored in a retrievable manner (R3) and the method ends. The method steps can be repeated for a second microscope 9.

[0100] Reference Figure 12 A version of the method for comparing two optical systems, in particular two microscopes 9, is described. A reference sample 1 is placed in a first microscope 9, the relevant sample points, in particular the desired test pattern 5, 6, 7 or 8, are sought and an image recording is prepared. For this purpose, the necessary parameters for the image recording must be known or stored (step V1). These parameters are, for example, the temporal resolution, the pixel resolution, the light intensity, the detector gain, the illumination time, the scanning speed and the spectral division. The image capture and the evaluation of the image data acquired also belong in step V1. The image capture can include all spatial dimensions (2D, 3D) and temporal profiles. The goal of the evaluation is, for example, to classify features as relevant. Such a feature is, for example, a captured intensity profile of a test pattern 5, 6, 7 or 8, in which at least one distance between maxima of the intensity is evaluated.

[0101] The evaluation can additionally or alternatively be based on the size of the regions of the substructure 5.1 to 8.1, 14.1 that are excited to emit luminescence (for example, using the full width at half maximum); on the size of the gaps between the excitation regions (for example, via the Rayleigh, Dawes, Sparrow criteria); on the uniformity of the excitation regions; on the temporal constancy or variation of the excitation regions. The evaluation can be based on criteria such as the Nyquist-Shannon theorem, the signal-to-noise ratio, etc.; on the contrast; on the resolution required. The reference sample 1 is removed from the first microscope 9.

[0102] 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 the evaluation result of the first microscope 9 should be compared with the evaluation result of the second microscope 9. If the answer is no (N), a comparison with a theoretical value is carried out in step V4. This can be, for example, the theoretically achievable resolution according to the Abbe criterion. The result of the comparison is output or the first microscope 9 is classified (R5) and the method ends.

[0103] On the other hand, if the comparison with the second microscope 9 in the decision step VE1 is positive (Y), the reference sample 1 is placed into the second microscope 9 and the measurements corresponding to V1 are carried out in step V2. The results (R2) are output and stored.

[0104] The system parameters of the first microscope 9 are applied to the second microscope 9 as far as possible. For example, the temporal resolution, the pixel resolution, the light intensity, the detector gain, the exposure time, the scanning speed and / or the spectral division can be adopted. If necessary, the parameters for image capture are adapted to the requirements of the second microscope 9. For example, when comparing a laser scanning microscope with a widefield microscope or a high-resolution SR microscope with a laser scanning microscope or a widefield microscope, adjustments can be necessary.

[0105] The results from the first microscope 9 from R1 are then compared with the results from the second microscope 9 from R2 in step V3. The result of V3 is output (R4) as a comparison value between the two microscopes 9. In a subsequent step V4, a comparison with the theoretical value takes place and the result is output in R5 and the method ends.

[0106] Reference signs

[0107] 1 reference sample

[0108] 2 carrier structure

[0109] 3 carrier material

[0110] 4 slide

[0111] 5 first structure / first test pattern

[0112] 5.1 substructure (of the first structure 5)

[0113] 5ax axial test pattern

[0114] 5.1ax axial substructure

[0115] 6 second structure / second test pattern

[0116] 6.1 substructure (of the second structure 6)

[0117] 7 third structure / third test pattern

[0118] 7.1 substructure (of the third structure 7)

[0119] 8 fourth structure

[0120] 8.1 substructure (of the fourth structure 8)

[0121] 9 microscope

[0122] 10 evaluation and control unit

[0123] 11 objective

[0124] 12 sample table

[0125] 13 driver

[0126] 14 fifth structure / fifth test pattern

[0127] 15 comparison unit

[0128] 16 base body

[0129] 17 machine-readable code

[0130] oA optical axis

[0131] BI image from scene I

[0132] B II1 first image from scene II

[0133] B II2 second image from scene II

[0134] Ki, Vi method steps; where i = 1 to 7

[0135] KEi, VEi decision steps, where i = 1 to 5

[0136] Ri result steps, where i = 1 to 3

[0137] S intersection point

Claims

1. A 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 is at least in its range excitable to emit luminescence, wherein the carrier material (3) having at least one two-dimensional and / or three-dimensional structure (5, 6, 7, 8, 14, 17) which is predetermined in terms of size and shape and which is composed of a plurality 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 the region of or around the two-dimensional and / or three-dimensional structure (5, 6, 7, 8, 14, 17) so as to be excitable to emit the luminescence; and - the respective substructure (5.1, 5.1ax, 6.1, 7.1, 8.1, 14.1) is formed laterally and / or axially as a region, as a point, as a three-dimensional structure or as a combination of at least two line segments, wherein the lateral substructures (5.1, 6.1, 7.1, 8.1) extend on or below the surface of the reference sample and substantially parallel thereto, while the axial substructures (5.1ax, 14.1) extend into the carrier material in the z direction or inclined to the z direction.

2. The reference sample (1) according to claim 1, characterized in that The carrier material (3) has regions doped with pairs of nitrogen vacancies, silicon vacancies, phosphorus vacancies, oxygen vacancies, sulfur vacancies, tin vacancies and / or manganese vacancies.

3. The reference sample (1) according to claim 1 or 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 segments which have a distance (di) between the respective line segments, wherein the distance (di) between the line segments changes stepwise with different pairs.

4. The reference sample (1) according to claim 3, characterized in that The distance (di) is selected from the range of 25 nm to 500 nm.

5. The reference sample (1) according to claim 1 or 2, characterized in that The axial substructure (5.1ax) comprises at least one pair of intersecting lines.

6. The reference sample (1) according to claim 1 or 2, characterized in that The lateral substructure (6.1) of the respective structure (6) is formed as a region excitable to emit luminescence.

7. The reference sample (1) according to claim 6, characterized in that The thickness of the lateral substructure (6.1) in the z direction is less than or equal to 50 nm.

8. The reference sample (1) according to claim 7, characterized in that The thickness is less than or equal to 25 nm.

9. The reference sample (1) according to claim 6, characterized in that The lateral substructure (6.1) is arranged alternately with regions not excitable to emit luminescence.

10. The reference sample (1) according to claim 1 or 2, characterized in that The substructure (6.1) is formed as a region not excitable to emit luminescence and is embedded in a matrix (16) excitable to emit luminescence.

11. The reference sample (1) according to claim 1, characterized in that The structure further comprises a substructure formed as a machine-readable code.

12. Use of a reference sample (1) according to any one of claims 1 to 11 for calibrating a microscope (9), comprising the following steps: - providing the reference sample (1) 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 introduced into the 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 result in a retrievable form.

13. A method for referencing at least two microscopes (9) to each other, comprising the following steps: - providing a reference sample (1) according to any one of claims 1 to 11 in the beam path of a first microscope (9), 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 introduced into the object field of the first 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 first microscope (9); - evaluating the acquired image data and storing the evaluation result as a first evaluation result; - providing the reference sample (1) in the beam path of a second microscope (9), 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 introduced into the object field of the second 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 second microscope (9); - evaluating the acquired image data and providing the evaluation result as a second evaluation result; and - comparing the first evaluation result and the second evaluation result to each other.

14. The method of claim 13, wherein, The first evaluation result and / or the second evaluation result are compared to an expected value by means of a comparison unit (15).

Images