DE102014205660B4 - Test specimen for the calibration of a Raman microscope - Google Patents

Abstract

Test specimen (10) for calibrating the position of a sample table of a Raman microscope with a substrate (12) made of a first material and a calibration structure (14) made of a second material different from the first material, at least either the first material or the second material is Raman active, characterized in that the calibration structure (14) has a thickness d of less than 30 nm.

Description

The invention relates to a test specimen for calibrating a sample table of a Raman microscope and a method for producing a test specimen for calibrating a sample table of a Raman microscope.

A Raman microscope is a microscope that uses the characteristic vibration vibrations in molecules for imaging, in that after excitation of the molecules by means of a laser, the photons emitted during the relaxation are detected and the Stokes lines are thus determined. In addition to the light-optical image, the Raman spectra of the sample are also recorded with a Raman microscope. A raster process is used so that the Raman spectra are spatially resolved. The examined area with the selected step size is moved line by point under the microscope objective. This process is called "mapping". The Raman microscope thus combines optical microscopy and Raman spectroscopy.

Raman microscopes are used today in particular to examine biological systems at the cellular level, possibly also in a time-resolved manner. In this way, images of living cells can be generated. It is also possible to use marker methods e.g. Understand transport processes, which is relevant for clinical research work, for example. Raman microscopy is a versatile measurement method for the non-destructive characterization of e.g. chemical sample composition, crystallinity and mechanical stress influences. Since spatially resolved information in the micrometer range is obtained by scanning the sample step by step under the microscope, the quality of the images obtained is crucially dependent on the scanning device.

Therefore, a Raman microscope, like any high-resolution scanning microscope, must be calibrated. In particular, the sample table must be calibrated to ensure that the desired position of the sample to be examined matches its actual position. A common method for calibrating microscopes is to use a so-called standard. Such a standard is a test specimen with precisely defined, measurable properties that can be traced back to the international system of units (SI) through a completely closed calibration chain. The test specimen is measured with a microscope and the measurement results obtained are compared with the data known from the test specimen and a calibration is carried out.

For the calibration of scanning devices on Raman microscopes, test specimens have hitherto generally been used which can also be used for other types of microscope. Known test specimens, e.g. for atomic force microscopes, scanning electron microscopes or fluorescence microscopes u. a. manufactured by Plano and NT-MDT. However, the available 1D line gratings with 120 nm deep notches do not show sufficient Raman contrast between the upper and lower plateaus and additionally lead to disruptive edge effects.

From the US 7 450 227 B2 a substrate for Raman spectroscopy is known which has a porous metal film. The structuring increases the Raman scattering signal by several orders of magnitude. The substrate or the sensors described there are not suitable for calibrating the spatial resolution of a Raman microscope.

In the US 2013/0 149 500 A1 describes a method for producing a nanostructured material that can be used to produce Raman samples. This substrate is also not suitable for the spatially resolved calibration of Raman microscopes.

The US 6 081 328 A describes another way to improve the Raman spectrum of a substance by applying a metal layer to the surface of the substance. The laser with which Raman spectroscopy is carried out is then directed onto the metallic layer so that the laser beam passes through this metallic layer. The Raman scattered radiation passes through the metallic layer again and is then detected. Such samples are also unsuitable for the spatially resolved calibration of Raman microscopes.

From the US 2013/0 242 297 A1 Another substrate for SERS (SERS = Surface Enhanced Raman Specostropy) investigations is known. The corresponding substrates have a base, a first layer of metal nanoparticles and a second layer of a plurality of metal nanoparticles which adhere to the surface of the metal nanoparticles of the first layer. Such a substrate is not suitable for the spatially resolved calibration of Raman microscopes.

Another method for producing a substrate is known from the US 7 864 312 B2 known. There, a semiconductor surface is exposed to short-term laser pulses, so that micrometer-sized structures are created on the surface. Such a substrate is also not suitable for the spatially resolved calibration of a Raman microscope.

Another substrate is from the US 2013/0 196 449 A1 known. This substrate has a signal-enhancing structure and an analyte receptor attached to this structure. Even with such a substrate, a spatially resolved calibration of a Raman microscope is not possible.

The object of the invention is therefore to provide a test specimen which enables increased accuracy in the calibration of a Raman microscope, the calibration being able to be carried out without additional equipment.

The invention achieves the object by means of a test specimen with a substrate made of a first material and a calibration structure applied thereon made of a second material different from the first material, at least either the first material or the second material being Raman-active and the calibration structure being one thickness d ₁ of less than 30 nm.

1. a. Aufbringen einer Schicht eines photosensitiven oder elektronenstrahlsensitiven Lacks auf einem Substrat, das aus einem ersten Material besteht,
2. b. Belichten der Lackschicht mit einem zur Kalibrierung eines Raman-Mikroskops geeigneten Muster,
3. c. Aufbringen einer Schicht einer Dicke d₁ von unter 30 nm eines von dem ersten Material verschiedenen Materials auf das Substrat, wobei das erste Material und/oder das zweite Material Raman-aktiv ist,
4. d. Entfernen der Schicht des photosensitiven Lacks .

The object is further achieved by a method for producing a Raman test specimen with the steps:

1. a. Applying a layer of a photosensitive or electron-beam-sensitive lacquer to a substrate which consists of a first material,
2. b. Exposing the lacquer layer with a pattern suitable for the calibration of a Raman microscope,
3. c. Apply a layer of a thickness d ₁ of less than 30 nm of a material different from the first material onto the substrate, the first material and / or the second material being Raman active,
4. d. Remove the layer of photosensitive paint.

Furthermore, the object is achieved by using a test body according to the invention for the calibration of a Raman microscope.

The invention is based on the finding that a calibration with higher accuracy can be achieved using the Raman spectrometer integrated in the Raman microscope. According to the invention, a test specimen consists of a substrate made of a first material and a calibration structure applied thereon made of a second material different from the first material, at least either the first material or the second material being Raman-active and the calibration structure being a thickness d ₁ of less than 30 nm. The test specimen is therefore not structured like known test specimens with regard to its topography or fluorescence, but with regard to local differences in the Raman activity. If the structure of the test specimen is known with sufficient accuracy, traceable calibration of the Raman microscope is possible.

A test specimen is understood in particular to mean a body whose structure is precisely known. For example, the structure can be measured with high precision and documented by a test certificate. A body that has a structure, but whose structure is not known with sufficient accuracy, is unsuitable as a test body. For a test specimen according to the invention for the calibration of a Raman microscope, the structure, i.e. in particular the dimensions of the structure layer in the x and y directions with an expanded measurement uncertainty of 1% (expansion factor k = 2) are known.

A Raman-active material is understood to mean, in particular, a material that has a polarizability that is variable during a molecular or lattice oscillation.

There is both the possibility that the first material and thus the substrate is Raman-active, and the second material and thus the calibration structure is Raman-inactive, as well as the reverse case that the first material and thus the substrate is Raman-inactive and the second material and thus the calibration structure is Raman active. The only decisive factor is that there is a sufficiently high contrast between the substrate and the calibration structure in the Raman image. This can also be the case if both the first and the second material are Raman active. The decisive factor is the contrast that results after adding the signals of both materials to the wave number considered for calibration.

A calibration structure is understood to mean a structure that is shaped and dimensioned in such a way that it is suitable for the calibration of the microscope. A suitable structure for calibration in one dimension has e.g. the shape of a measuring tape including the associated scale. In other words, the calibration structure is suitable for measuring quantities such as distances, angles and the like. Ä. to compare with known sizes, which are generally known based on a previous measurement of the test specimen. The calibration structure is generally largely or completely opaque for visible light and generally for excitation radiation emitted by the Raman microscope. SERS substrates often have similar structures, but are still not suitable for calibration because the dimensions are not of the required size.

It has been found that a pronounced topography is disadvantageous for such a test specimen, since edge effects in the form of intensified intensities can result at the edges of the calibration structure. It is therefore necessary that the calibration structure is as thin as possible. A thin structure is understood to mean a structure whose thickness is less than 30 nm, preferably less than 20 nm. The result is an almost topography-free test specimen.

A high contrast is desirable, that is to say a large ratio between the Raman activity of the regions covered by the calibration structure and the Raman activity of the uncovered regions of the substrate. The contrast can also arise from differences in the wavelength of the radiation emitted as Raman radiation.

The contrast can be influenced on the one hand by the choice of material and on the other hand by the layer thickness of the calibration structure. A thicker layer creates better shading of the underlying substrate, so it leads to less Raman activity in the area of the calibration structure. At the same time, the edge effects also increase with the layer thickness, as described above, so that a compromise between the desired high contrast and undesired edge effects must be found here. The calibration structure therefore advantageously has a thickness between 10 and 25 nm, preferably between 15 and 20 nm. In experiments carried out by the inventors, test specimens with a layer thickness of the calibration structure between 15 and 20 nm have been found to be optimal.

An advantage of a test specimen according to the invention is that it permits calibration of the adjustment device of the microscope stage in point-by-point or line-by-line scanning on the basis of the Raman emission. Furthermore, the point spread function of the optical resolution in point-by-point or line-by-line scanning can be determined using the Raman signal.

The test specimen according to the invention has calibration structures which have no edge artifacts and are therefore almost topography-free. It is possible to produce a test specimen which has periodic structures which, with a small thickness of the calibration structure, have a sufficient Raman contrast even with small pitches in order to calibrate small areas very precisely. Periodic structures can also be present on the test specimen which are large enough to calibrate long ranges.

The different patterns of the calibration structure can be present in different sizes on the test specimen in order to cover all common combinations of excitation wavelengths and objective as well as different pitches and different circle diameters.

By using two-dimensional, orthogonal structures, both main axes can be calibrated simultaneously and distortions in the image made visible. It is possible to trace back to the meter with the help of reference analytics (AFM, REM). Furthermore, the test specimen can be wetted with a drop of immersion liquid and cleaned again, since it can be made resistant to organic solvents.

A problem in the manufacture of a test specimen according to the invention can be poor adhesion of the calibration structure to the substrate. A further development of the invention therefore provides that an adhesion promoter is located between the substrate and the calibration structure, the thickness of a layer formed by the adhesion promoter preferably being less than 5 nm. Such an adhesion promoter can e.g. Be titanium. In principle, however, any other material is also suitable which has good adhesion to the substrate and on which the calibration structure can be applied.

Semimetals such as silicon and germanium are preferably conceivable as the substrate, the Raman activity of germanium being significantly lower than that of silicon. Quartz is a suitable non-metallic material for a Raman-active substrate. Ramaninactive substrates can consist of metals with the crystal systems fcc, hcp and bcc (gold, palladium, titanium or platinum). These metals are also suitable Raman inactive materials for the structural layer and are able to “shade” the underlying substrate, so that there is a high contrast in the Raman image. An alloy of gold and palladium is particularly suitable as the material of the calibration structure. The ratio of gold to palladium can e.g. are in the range between 40:60 and 70:30. In other words, the calibration structure contains 40 to 70% gold and 60 to 30% palladium. In addition to silicon and germanium, a suitable material for a Raman-active structure layer or calibration structure is e.g. Graphene, a one-layer modification of carbon. So e.g. If a combination of graphene on silicon is selected, the calibration structure can be seen in the Raman image at the wavenumber used for the calibration and the substrate remains dark, whereas e.g. Gold on silicon provides the inverse picture. Graphene is also characterized by very good adhesion even without an adhesion promoter on the substrate.

In order to enable a comprehensive calibration, the calibration structure can use several of the The following patterns include: checkerboard, dot grid, line grid, point scattering centers, large areas with sharp edges. Any combination is possible depending on the specific requirements.

A pattern is any regular structure that allows calibration. As a rule, a pattern repeats itself after a certain spatial period. However, this is not absolutely necessary, so a "large area with sharp edges" can also be created from any image of an object or e.g. consist of a character.

Two-dimensional, orthogonal structures are regarded as the “checkerboard pattern”. These can be made in different sizes, but are at least 200 µm × 200 µm in size. Such checkerboard-like structures are suitable for calibrating both lateral main axes at the same time without the need to rotate the sample, that is to say the test specimen. It has been found that different calibration factors for the table can result from the calibration of a single main axis in the line mapping method than with a two-dimensional mapping.

If the calibration structure has a checkerboard arrangement, this can continue to serve for the simultaneous assessment of the uniformity of the line shift, which would not be possible by means of a calibration structure which only has point grids. Such a two-dimensional structure does not necessarily have to be orthogonal and a checkerboard pattern, e.g. diamond-like or hexagonal structures.

A “dashed grating” is understood to mean a periodic, one-dimensional structure which can have different distances between the individual parts of the structure, so-called pitches. The structures are of course not one-dimensional in the strictly geometrical sense, but have a width of at least 200 µm. The geometrical structure of the grating allows the measurement point to be recorded over the entire adjustment range of the microscope stage. Due to the increased contrast compared to the two-dimensional structures, the dashed grids also enable 1D calibration for very small pitches in the order of magnitude close to the optical resolution.

The structures are advantageously produced multiple times with different pitches. A large pitch of e.g. 4 µm to cover a large calibration range with a step size of e.g. 1 µm to ensure a minimum scanning corresponding to the Nyquist theorem. A relatively small pitch of 0.8 µm provides a sufficient number of periods for high-resolution calibration of small areas with a step size of e.g. 0.1 µm safe. Another pitch of e.g. 2 µm makes it possible to cover different excitation wavelengths or lens combinations and step sizes.

Individual point scatter centers can be present as further features of the calibration structure. These can e.g. Have diameters of 100 nm, 200 nm and / or 400 nm. With the aid of such point scattering centers, the detected point spread function and thus the optical resolution can then be determined. There may also be double point scattering centers. The diameters can correspond to those of the individual point scattering centers; In the case of the 100 nm point scattering centers, the center distance is expediently four times (400 nm), otherwise twice (400 nm, 800 nm) the diameter. Using such double point scattering centers, the optical resolution e.g. validate according to the Rayleigh criterion.

The point scattering centers can be used to determine the profile of the excitation radiation. The laser beam of a Raman microscope usually has a profile that essentially corresponds to a Gaussian curve; the radiation intensity as a function of the distance from the center of the optimally circular laser beam thus follows a Gaussian function, the maximum of which lies in the center of the laser beam. However, this theoretical Gaussian profile is influenced by the other optical components of the structure, so that the Gaussian profile can only be an approximation for a real structure. With the aid of point scattering centers on the test specimen, the real beam profile can be determined and used for the unfolding of the image data obtained. This leads to a significant increase in the accuracy of the method.

Large areas with straight, sharp edges allow the half-width of the detected point spread function to be determined. For this purpose, the edge can be scanned using a linemapping method and the intensity function obtained can be fitted as a Gaussian error function (erf). A “large area” is understood to mean a homogeneous, uninterrupted area which has an edge length which corresponds to at least five times a period of the regular structures used, which has an edge length of 5 μm × 5 μm, for example. A “sharp edge” is understood to mean that the transition area between the structural material and the uncoated substrate is as small as possible, ideally zero. Accordingly, all calibration structures used have sharp edges. The layer thickness of the calibration structure preferably increases over a distance of less than 50 nm from less than 5% to at least 95% of the nominal layer thickness. Under a straight edge there will be one understood maximum deviation from a geometric straight line from 50 nm to 1 µm.

It is expedient to make the calibration structure redundant on the substrate, so that the structure preferably comprises a plurality of different patterns and each pattern is present at least 10 times identically. It is less complex to apply a plurality of possibly identical structures to a single test specimen than to produce a plurality of test specimens, on each of which only a single structure is applied. With the redundancy mentioned, it can thus be avoided with little effort that the entire test specimen becomes unusable in the event of a faulty production of a single structure. Instead, it can be determined which structures were produced without errors, so that only these are then used for calibration. It is then not necessary to use another test specimen, instead an identical structure applied in another area of the test specimen is simply used for calibration.

A test specimen according to the invention can be produced using the following method: coated with an electron beam sensitive lacquer or PMMA (polymethyl methacrylate) and then exposed using proximity corrected electron beam lithography. A photosensitive or electron beam sensitive lacquer is understood to mean any substance that is suitable for the production of a mask, in addition to PMMA e.g. also other electron-sensitive coatings such as ZEP-520 from Zeonrex Electronic Chemicals. The desired structures are transferred to the substrate. The exposed structure is then developed. A resist mask is obtained, into which the exposed structure has been transferred. A thin layer of titanium and then a gold-palladium alloy are deposited on this resist mask in a thermal evaporator, the alloy being e.g. Contains 60% gold and 40% palladium. As an alternative to a thermal resistance evaporator, an electron beam evaporator can also be used. It is important that the material is separated from a point-like source.

Both the titanium layer and the working layer, i.e. the layer that later forms the calibration structure, are thin. Of course, the titanium layer is expediently thinner than the working layer, since above a certain thickness no better adhesion is achieved.

Possible materials for the calibration structure differ mainly in their fine grain and in their behavior during the so-called lift-off, i.e. the removal of the paint mask. The fine grain is important in order to produce highly precise, smallest structures and thus determines the smallest possible structure size and therefore indirectly also the accuracy of the calibration. It is also important that the material adheres well to the substrate during the lift-off, so that no defective structures are produced. An alloy containing 60% by weight of gold and 40% by weight of palladium has proven to be advantageous. With such an alloy, structures with sizes down to 100 nm can be produced with distances between the structures in the range down to 10 nm.

The paint mask is then removed using a solvent. The exposed pattern is now transferred to the surface of the wafer as a metallic structure. The structures produced in this way, i.e. the entire material applied to the wafer, then have a thickness of e.g. approx. 23 nm. This is made up of a thin adhesion promoter layer, which consists of an approx. 3 nm thick titanium layer, and a working layer, which consists of gold and palladium and is approx. 20 nm thick.

• 1 eine schematische Darstellung eines erfindungsgemäßen Prüfkörpers in Draufsicht,
• 2 eine schematische Darstellung eines Layouts einer Kalibrierstruktur,
• 3 eine vergrößerte Darstellung eines einzelnen Bereichs einer Kalibrierstruktur,
• 4 eine vergrößerte Darstellung eines weiteren einzelnen Bereichs einer Kalibrierstruktur, und
• 5 eine schematische Darstellung eines Schnitts durch einen erfindungsgemäßen Prüfkörper.

The invention will be explained in more detail below with reference to the attached figures. Show it

• 1 1 shows a schematic illustration of a test specimen according to the invention in plan view,
• 2 1 shows a schematic representation of a layout of a calibration structure,
• 3 an enlarged view of a single area of a calibration structure,
• 4 an enlarged representation of a further individual area of a calibration structure, and
• 5 is a schematic representation of a section through a test specimen according to the invention.

1 shows an embodiment of a test body according to the invention in plan view. The test specimen 10 consists of a silicon chip as a substrate 12 as well as the applied calibration structures 14.1 . 14.2 , .... The chip has an edge length of approx. 3 cm x 3 cm. Other edge lengths of, for example, 2 cm × 2 cm, 4 cm × 4 cm are, however, just like rectangular substrates 12 , for example with an edge length of 3 cm × 4 cm, conceivable and suitable. The calibration structures shown as rectangles can be seen 14.1 . 14.2 , .... There are a total of 20 identical calibration structures 14.1 . 14.2 , ... on the silicon chip 1 applied. A higher or lower number, e.g. 5 × 5 = 25, 4 × 4 = 16 or 10 × 10 = 100 calibration structures 14.1 . 14.2 , ... is also possible. Each of these calibration structures 14.1 . 14.2 , ... is arranged in a rectangular field, which Edge lengths of a = 4.5 mm and b = 3.5 mm. Such an area of less than 20 mm ² in this case is sufficient to accommodate all the necessary patterns. A test specimen 10 the size of such a field would be difficult to handle on the one hand, and on the other hand, with a substrate with an edge length of at least 2 cm, it is possible to apply a drop of an immersion liquid, for example from oil, to the test specimen and also to remove it again. During cleaning, the test specimen can be gripped and completely cleaned in a bath made of organic solvents. Therefore, a larger chip is conveniently used as a substrate 12 is chosen. A plurality of identical calibration structures can then be placed on this 14.1 . 14.2 , ... are applied, which greatly reduces the reject rate.

2 shows a sketch of an enlarged representation of a calibration structure 14.1 . 14.2 , .... In such a calibration structure 14.1 . 14.2 , ... out 1 Rectangle containing are the in 2 shown 12 substructures or patterns 18.1 . 18.2 , ... available. The following can be seen as a pattern in column A: CV Raman as a pattern with large areas and sharp edges, squares with sharp edges, single and double point scattering centers; in column B: two-dimensional orthogonal structures in the form of checkerboard patterns, in column C: point grids of different distances and in column D: one-dimensional structures in the form of grids.

The patterns are available in different sizes. The diameters of the point scattering centers vary 18.2 . 18.3 as well as the distances between the points 18.4 . 18.5 . 18.6 , the lines 18.7 . 18.8 . 18.9 and the individual fields of the checkerboard pattern 18:10 . 18:11 . 18:12 , Column B gives an example of the periodic structures, which correspond to the pitches. In the upper chess board 18:10 the period is 4 µm, in the middle chessboard 18:11 2 µm and in the lower chessboard 18:12 0.8 µm. A single field of chess boards 18:10 . 18:11 . 18:12 has an edge length that corresponds to half the period.

3 shows a single calibration field in the lower area 20.1 as a section of a calibration structure 14.2 , which is shown again schematically as an overview in the upper area. The other calibration fields shown in the upper area 20.2 . 20.3 , ... can have other structures, such as those in 2 shown are included. The one in the calibration field 20.1 contained circular and square point scattering centers 18.2 are available in different dimensions, the edge lengths a vary from 0.1 µm (bottom) to 3.2 µm (top). The edge length I of the calibration field 20.1 is 400 µm × 400 µm.

4 shows a single calibration field in the lower area 20.2 as a section of a calibration structure 14.2 , which is shown again schematically as an overview in the upper area. The other calibration fields shown in the upper area 20.1 . 20.3 , ... can have other structures, such as those in 2 shown are included. The calibration field shown 20.2 has a checkerboard structure 18:11 as a two-dimensional, orthogonal structure and is suitable for the calibration of the lateral main axes of the sample. The period a is 0.8 µm in the case shown, but can also be, for example, 2 µm or 4 µm

5 shows a section through a region of a test specimen according to the invention 10 , The representation is not to scale. It can be seen that the substrate made of silicon 12 accounts for the largest part of the test specimen. The silicon substrate is in some areas 12 not covered by another layer, so that the excitation radiation incident from above 28 of the Raman microscope can penetrate freely to the substrate. The high Raman activity of the silicon is determined at the corresponding points based on the Raman scattering 30 measured. In the picture on the left and right are a thin layer 16 the thick d ₂ of titanium and a layer 22 the thick d ₁ a gold-palladium alloy. Through these layers 16 . 22 becomes the silicon substrate 12 covered so that neither excitation radiation 28 unhindered to the substrate 12 can penetrate the possibly resulting photons of the weakened Raman scattering belonging to the Stokes line 32 can easily get back to the microscope objective and from there to the spectrometer. The edges 24 . 26 are perpendicular to a good approximation. A highly precise execution of the structure is necessary in order to enable an exact calibration.

The top layer or working layer 22 made of gold and palladium is Raman-inactive, so that there is a high contrast in the Raman image between the areas that are coated and the uncoated areas. The areas that are coated with titanium and the gold-palladium alloy form the calibration structure 14 , The total thickness of the calibration structure 14 is the sum of the two thicknesses d ₁ the gold-palladium alloy and the thickness d ₂ of titanium.

LIST OF REFERENCE NUMBERS

10

specimen

12

substratum

14

calibration structure

16

bonding agent

18

template

20

calibration field

22

work shift

24

edge

26

edge

28

incident radiation

30

Raman scattering

32

weakened Raman scatter

a

Diameter, period

d ₁

Working layer thickness

d ₂

Adhesion layer thickness

I

edge length

Claims (10)

Test specimen (10) for calibrating the position of a sample table of a Raman microscope with a substrate (12) made of a first material and a calibration structure (14) made of a second material different from the first material, at least either the first material or the second material is Raman active, characterized in that the calibration structure (14) has a thickness d ₁ of less than 30 nm. Test specimen (10) Claim 1 , characterized in that the calibration structure (14) has a thickness d ₁ between 10 and 25 nm, preferably between 15 and 20 nm. Test specimen (10) Claim 1 or 2 , characterized in that there is an adhesion promoter (16) between the substrate (12) and the calibration structure (14), the thickness d _{2 of} a layer formed by the adhesion promoter (16) preferably being less than 5 nm. Test specimen (10) Claim 1 or 2 , characterized in that the calibration structure (14) consists of a Raman-active material. Test specimen (10) according to one of the Claims 1 to 3 , characterized in that the calibration structure (14) consists of 40 to 70 wt .-% of gold and 30 to 60 wt .-% of palladium. Test specimen (10) according to one of the preceding claims, characterized in that the calibration structure comprises a plurality of point scattering centers. Test specimen (10) according to one of the preceding claims, characterized in that the calibration structure (14) comprises several of the following patterns: checkerboard pattern, point grid, line grid, point scattering centers, large areas with sharp edges. Test specimen (10) according to one of the preceding claims, characterized in that the calibration structure (14) on the substrate (12) is of redundant design, the calibration structure (14) preferably comprising a plurality of different patterns and each pattern being at least 10 times identical is available. Method for producing a test specimen (10) for calibrating the position of a sample table of a Raman microscope, comprising the steps: a. Applying a layer of a photosensitive or electron beam sensitive lacquer to a substrate (12) which consists of a first material b. Exposing the lacquer layer with a pattern suitable for calibration of a Raman microscope using electron beam lithography c. Applying a layer of a thickness d ₁ of less than 30 nm of a second material different from the first material to the substrate, the first material and / or the second material being Raman active d. Removing the layer of the electron beam sensitive lacquer Use of a test specimen (10) according to one of the Claims 1 - 8th for the calibration of a Raman microscope.

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