Method and assembly for determining an offset value of an imaging optical system, and computer program

Abstract

The invention relates to a method 100 for determining an offset value for offset correction of an immersion objective lens of an imaging optical system. The method comprises the following steps: imaging 101, 110, 120 of an object using a reference objective lens and an immersion objective lens of the optical system without immersion, and ascertaining 103, 131, 141 offset values between the reference objective lens and the immersion objective lens by means of the imaging of the object in the presence of immersion. The invention further relates to a component 200 and to a computer program.

Description

[0001] The present invention relates to a method for determining the immersion objective offset value of an imaging optical system, a component having an imaging optical system, and a computer program.

[0002] In order to obtain a micrograph of a high magnification of an object to be imaged, such as a sample or a specimen, it is common for the user to first use an objective lens with a small magnification to find and position the sample as efficiently and collision-free as possible by taking advantage of a large depth of field and a large image field. Then, the objective lens with a high magnification is replaced. In addition, due to the different tolerances of the microscope components, especially the objective lenses, when the objective lens is replaced, a large deviation may occur in the focus orientation. This may require relatively strong refocusing, which may lead to an inefficient working process. In the case of automatic focusing being carried out automatically, there is also a risk that the focus of the sample is lost and the working process is disturbed.

[0003] Here, a remedial measure can be to implement the installation of washers with different thicknesses, and these washers are used to appropriately correct the position of the objective lens. However, such manual correction is time-consuming and can only be performed by professionals at the position of the optical system.

[0004] For this reason, in a Z-motorized microscope, a parfocal position correction can be implemented, hereinafter also referred to as parfocality correction. For this purpose, based on the known difference in the focus position, when a specific objective lens is pivoted, its position relative to the sample can be corrected, for example, by means of a microscope mount, so that the focus orientation is maintained. A corresponding method is described in DE 10 2008 063 799 A1.

[0005] In this regard, the term "parfocality" represents the following characteristic of an imaging optical system, that is, the once clearly set position of the object to be imaged continues to be clearly imaged even when the system changes, such as when the objective lens is replaced. For example, in the case of a fully parfocal optical microscope, the exact same plane of the object to be imaged is clearly presented by all objective lenses without having to re-correct the clarity when the objective lens is replaced. However, in an actual microscope system, a parfocal position correction or parfocality correction needs to be carried out to obtain the best focus of the target structure again when an optical component, such as an objective lens, is replaced, and the best focus may be lost, for example, due to material tolerances.

[0006] For this purpose, usually the user or the operator has to carry out refocusing, that is, adapt the position of the sample or the imaging component so as to image the target structure clearly onto the image plane again. Such a focus misalignment may be an inherent characteristic of the optical system. In a high-resolution microscope system, the depth of field is in the range of a few micrometers and the manufacturing tolerances are already sufficient to no longer accurately hit the focus orientation after the component is replaced.

[0007] Such misalignments can be corrected automatically in a motorized optical system. However, it is absolutely necessary to know the parfocality offset value required for performing parfocality correction.

[0008] However, the processes hitherto used to determine the required parfocality offset value are very complex and can only be performed partially manually. In particular, immersion objectives result in a significantly increased time consumption or a significantly longer calibration time here, since the immersion liquid has to be applied and then completely removed from the sample again. Additionally, the application of the immersion liquid is mostly associated with the removal of the sample, which again results in inaccuracies when determining the Z correction to be performed for parfocality during the process.

[0009] Furthermore, attention has to be paid to the order of the objectives since one proceeds from the objective with the highest resolution to the objective with the lowest resolution. Since this process has to be performed manually by the user, it is often error-prone. Additionally, the user always has to recalibrate all objectives since, according to the prior art, there is no solution for performing the correction for only one objective. Finally, this means that parfocality correction according to the prior art should only be performed by experienced and microscope-familiar users. This results in a significant limitation of user-friendliness and the efficiency of the microscopy process.

[0010] In summary, the methods hitherto used to perform parfocality correction mostly cannot be performed automatically or at least not fully automatically, but rather manually on the mount and only in combination with the respective eyepiece. In particular, due to the large number of method steps, the failure susceptibility is high. Thus, parfocality correction can only be achieved with difficulty, especially for inexperienced users. Additionally, the known methods are very time-consuming since all objectives always have to be recalibrated throughout the process. Furthermore, the parfocality correction of immersion objectives is extremely time-consuming due to the use of the immersion liquid.

[0011] In addition to the deviation in parfocality, a deviation in concentricity also occurs when changing the objective. Thus, in the case of changing the objective, in addition to parfocality correction, concentricity correction can also be meaningful. In this context, the term "concentricity" denotes the following property of the imaging optical system, namely that when the system changes, for example when changing the objective, the center point of the image remains at the image center and does not shift laterally. Concentric position correction or concentricity correction correspondingly enables achieving the best possible concentricity.

[0012] Against this background, the object of the present invention is to provide a method and a component by means of which the above-mentioned disadvantages can be at least partially avoided. It may be desirable to simplify the determination of the offset value, which can be performed on the one hand with little time and effort and on the other hand with high precision, preferably also by less trained users and at least partially automatically.

[0013] This object is achieved by the subject matter of the independent claims. The dependent claims relate to implementation variants of these solutions according to the invention.

[0014] The basic idea of the invention is that even in the absence of an immersion liquid, an immersion objective will achieve an optical imaging, by means of which an offset value can be determined. For example, the optical imaging generated in the absence of an immersion liquid can allow the focal position of the pivoted immersion objective relative to the sample structure or the target structure to be determined experimentally. Even if the obtained resolution is reduced and aberrations occur, the focal position can be determined with an accuracy of a few micrometers.

[0015] This can be carried out automatically by evaluating the images from a z-stack based on the contrast and / or resolution of the z-stack. In other words, the contrast and / or resolution of a plurality of images recorded at different distances between the objective and the sample in the z-direction (i.e., in the direction of the optical axis) can be analyzed and used to subsequently determine a correction value for performing parfocality correction.

[0016] Here, in a simple case, the user's sample can be used. Alternatively or additionally, at least one reference sample can be used, the structure of which is known and can possibly be optimized in terms of robustness and accuracy by cooperating with the evaluation of the image stack. However, if available, a hardware autofocus system (e.g., based on reflection, using propagation time measurement and / or based on triangulation) or an image-based, computer-implemented autofocus method can also be used to determine the position of the sample surface relative to the objective, as long as the detection principle provides reliable position values both in the presence and absence of an immersion liquid.

[0017] The basic idea described allows the offset value to be determined even in the absence of introducing the immersion liquid at the immersion objective, in particular the parfocality offset value that enables parfocality position correction relative to another objective mounted in the objective turret. Additionally or alternatively, concentric position correction can also be achieved by determining the corresponding eccentricity offset value.

[0018] The misalignment effect in the axial direction (i.e., in the z-direction) caused by the optical effect of the "lack of" immersion liquid can be determined theoretically, but can also be determined experimentally. Thus, a parfocality correction value can be determined, which can be used together with the introduction of the immersion liquid to correct the axial position of the focal position in the sample after changing between objectives. For concentric correction, it can be assumed with a good approximation that the immersion liquid, as an isotropic medium, does not cause misalignment (i.e., displacement) in the lateral direction.

[0019] A first aspect of the invention relates to a method for determining an offset value for offset correction of an immersion objective of an imaging optical system.

[0020] The method can be implemented, for example, by a computer using a data processing system, that is, at least one method step can be implemented using a computer program, preferably multiple or all method steps. There is a possibility of performing the method fully automatically, that is, without user interaction.

[0021] The imaging optical system can be an optical microscope, which, for example, provides different objective lenses with different magnifications or contrast selections on an objective lens turret. The optical microscope can be designed, for example, as an upright microscope or an inverted microscope. The imaging optical system has at least two objective lenses, at least one of which is designed as an immersion objective lens, that is, for use with an immersion liquid. All objective lenses can also be designed as immersion objective lenses. For example, water, glycerol, an oil mixture, oil, or silicone oil can be used as the immersion liquid, where the present invention is not limited to a specific immersion liquid.

[0022] The proposed method proposes that first, the reference objective lens and the immersion objective lens of the optical system are used to image the object without immersion respectively.

[0023] Then, the offset values of the offset between the reference objective lens and the immersion objective lens are obtained by means of or based on the imaging of the object without immersion, and these offset values represent the offset in the case of having immersion. In other words, the reference objective lens and the immersion objective lens are used to obtain the offset values without immersion, but the offset values of the offset are obtained in the case of having immersion.

[0024] Optionally, the basic offset values of the offset between the reference objective lens and the immersion objective lens (representing the offset without immersion) can be obtained in an intermediate step, and these basic offset values can then be used as the basis for obtaining the offset values. This means that the offset values can be obtained based on the basic offset values or directly or indirectly considering the basic offset values.

[0025] Here, in a simple case, the obtained basic offset values can be equal to the actual offset values. Thus, offset correction with sufficient accuracy can be achieved in many application cases in a very simple way and method.

[0026] Alternatively, the basic offset values can be converted into offset values by means of one or more corrections, for example, by using one or more correction values (especially correction values for the immersion). More accurate offset correction can be achieved by correcting the missing immersion during the imaging of the object.

[0027] Then the offset values can be used to perform offset correction, for example, to compensate for parfocality offset. The basic offset values and / or the offset values can also be stored for later use.

[0028] The reference objective lens can be an air objective lens or, equally, an immersion objective lens. If the imaging optical system has more than two objective lenses, one of the objective lenses can be used as the reference objective lens to determine the offset values for offset correction of the other multiple objective lenses.

[0029] Alternatively, the characteristics of the objective lens (which can be used as the reference objective lens) can be changed, i.e., the first reference objective lens is used to determine the offset value of the second objective lens. After this determination process, the second objective lens is used as the new reference objective lens to determine and, if necessary, correct the offset value of the third objective lens, etc.

[0030] A combination of these two process methods is also possible, i.e., for example, first a certain reference objective lens can be used to determine the offset values of the other multiple objective lenses, and then one of these other objective lenses is used as the new reference objective lens. The determination of the objective lens as the reference objective lens is freely determined, which defines which objective lens is used as the mathematical reference. Advantageously, during the measurement process, the basic offset values or the parameters on which they are based, such as the focal position of the target structure, are determined for all objective lenses without immersion. The basic offset value between two objective lenses, such as the parfocality basic offset value, can be directly determined based on the two measurement results assigned to the objective lenses without immersion, such as the obtained focal positions.

[0031] If an objective lens is subsequently inserted or replaced, this is sufficient to perform the proposed measurement process for the new objective lens and the to-be-defined reference objective lens, i.e., to determine the measurement parameter (such as the focal position) of the new objective lens without immersion on which the basic offset value is based and thus to determine the basic offset value. The new objective lens can be referenced by other objective lenses through the measurement of the reference objective lens.

[0032] According to the proposed method, it is possible to determine the offset values of all objective lenses without immersion. In this regard, the main discovery of the present inventor is that for an immersion objective lens, although it is designed to be used with an immersion liquid, the offset of the immersion objective lens can be determined accurately enough even without immersion to perform offset correction.

[0033] The proposed method advantageously enables offset correction in an imaging optical system with multiple objective lenses, especially in an optical microscope. This method can be executed with the same process flow regardless of the coverage of the immersion objective lens. This method can also be used for OEM systems because it is not necessarily required to use product-specific software. Therefore, this method can be used generally.

[0034] The proposed method provides the possibility of software-controlled and automated solution of motorized offset correction even in immersion objectives without performing automatic immersion or generally without applying immersion liquid. The method is no longer only capable of achieving compensations that have to be defined manually, but can implement workflows that can run without manual intervention by the user. In other words, the method can also be performed by less trained users or even automated without compromising accuracy.

[0035] This also relates, for example, to the sample carrier or the reference carrier, which are no longer contaminated by the immersion medium when calibrating the immersion objective. Cleaning is dispensed with and reliable reusability is ensured. Since the use of immersion liquid is dispensed with, the method also offers a significant time saving compared to the methods used hitherto for performing offset correction. In addition, user acceptance can be increased, i.e., the user may be more willing to perform the necessary offset correction, which can subsequently achieve more correct imaging results.

[0036] As long as the reference objective remains part of the optical system, i.e., remains mounted in the optical system, then in the case of replacing one or more objectives at other positions, for example, in the case of replacing the 3rd, 4th or 5th objective in addition to the above-mentioned reference objective and immersion objective, it may be sufficient to determine the offset values only for the newly introduced one or more objectives and to further use the other values.

[0037] Here it may be advantageous to compensate for the misalignment caused by the lack of immersion by means of the previously determined offset values, so that only the remaining deviations, for example caused by manufacturing tolerances, have to be measured and corrected. In the case of parfocality correction, the focal position can thus be reached more quickly, so that the method can be performed more quickly overall. In addition, the offset values can be determined more robustly and with higher precision because a basic correction has already been carried out.

[0038] According to different implementation variants, offset values can be determined for performing parfocality correction and / or concentricity correction.

[0039] In particular, parfocality correction, but also concentricity correction, enables high-quality optical imaging and simplifies possible subsequent image processing and image analysis.

[0040] Determining the offset values for performing parfocality correction can include, for example, the steps mentioned below:

[0041] Determine the focal position of the reference objective of the imaging optical system without immersion liquid, determine the focal position of the immersion objective without immersion liquid, and determine the parfocality offset value of the parfocality offset between the immersion objective and the reference objective based on the determined focal positions and at least one parfocality correction value. Here, the determination of the focal position can be performed based on the imaging of an object generated without immersion liquid using the reference objective or the immersion objective.

[0042] The determined parfocality offset value can optionally be stored for later use.

[0043] Optionally, the method can include compensating for the parfocality offset between the immersion objective and the reference objective by means of the determined parfocality offset value. Compensation for the parfocality offset can be particularly achieved when changing between the reference objective and the immersion objective (operating with immersion liquid) or in this regard.

[0044] The above- and below-mentioned method steps that are optionally mentioned can be implemented in the listed order, but can also be implemented in a different order or completely or partially overlapping in time if necessary. For example, it is also possible to first determine the focal position of the immersion objective and then determine the focal position of the reference objective.

[0045] According to the proposed method, it is possible to determine the focal positions of all objectives without immersion liquid. In this regard, the main finding of the present inventors is that for an immersion objective, although it is designed to be used with an immersion liquid, the focal position of the immersion objective can be determined accurately enough even without immersion liquid to perform the parfocality correction according to the proposed method.

[0046] Based on the determined focal positions of the reference objective and the immersion objective, the value of the actual focal position and thus the value of the parfocality offset (parfocality offset value) can be determined taking into account the parfocality correction value.

[0047] The parfocality correction value is used to correct for the missing immersion liquid when determining the focal position of the immersion objective and, if necessary, for the missing immersion liquid when determining the focal position of the reference objective (if the reference objective is also an immersion objective). In other words, the parfocality correction value can be regarded as an immersion correction value for correcting the parfocality offset. Therefore, the parfocality correction value between the objectives can be divided among the individual objectives, and it holds that: c ij = c + c i . It is clear from the definition that the value c i of the objective without immersion liquid is equal to zero.

[0048] One or more parfocality correction values can be determined once beforehand and stored in the proposed method for later application or, in other words, called from a storage unit within the scope of the proposed method. Additionally, there is the possibility, for example, of parameterizing the determined parfocality correction values in a computing unit with respect to correlations with environmental parameters such as temperature, carrier substrate thickness, etc. and deriving parfocality correction values for specific environmental conditions, as will be explained in more detail below.

[0049] "Determining a parfocality offset value based on the determined focus position" means that the determined focus position is considered directly or indirectly when determining the parfocality offset value. For example, the determined focus position can be corrected with the parfocality correction values of the respective objective lenses, and then the corrected focus position can be used to determine the parfocality offset values between these objective lenses.

[0050] Alternatively, it is possible to first determine a parfocality basic offset value of the parfocality basic offset between an immersion objective lens and a reference objective lens based on the focus position determined without immersion liquid, and then correct this parfocality basic offset value with the parfocality correction values. In this case, the parfocality offset value is determined based on the parfocality basic offset value and the parfocality correction values. To avoid errors when converting the parfocality basic offset value into the parfocality offset value, it may be advantageous if the reference objective lens is not an immersion objective lens. Since the measurement error for determining the focus is related to the reference objective lens, it may also be advantageous to select an objective lens with a low depth of field, i.e., with a high numerical aperture, as the reference objective lens.

[0051] Determining the parfocality basic offset value has the advantage that the associated offset values between the objective lenses caused by tolerances are determined and thus the bias values for the respective focus orientations are eliminated.

[0052] In a further, optional method step, after applying the immersion liquid to the immersion objective lens, the parfocality offset between the immersion objective lens and the reference objective lens can subsequently be compensated for by means of the determined parfocality offset value. This compensation can be achieved by suitable measures, such as moving or displacing the focusing drive of the immersion objective lens, moving the stage / holder, e.g., a microscope or the stage and / or the z-piezo holder, in the z direction, moving the immersion objective lens relative to the object to be imaged, or moving one or more correction lenses and / or motorized correction rings of the objective lens.

[0053] The determined parfocality offset values can be stored for later use, such that, without having to redetermine the focus position, the determined parfocality offset values can be used for subsequent reuse with this specific objective lens combination. In other words, in the case of replacing the objective lenses again, i.e., when reusing a specific objective lens, the parfocality offset values determined once can be directly used to compensate for the parfocality offset.

[0054] The proposed method advantageously enables parfocality correction in an imaging optical system with multiple objective lenses, especially in an optical microscope. This method can be executed with the same process flow regardless of the immersion of the immersion objective lens. This method can also be used in OEM systems because it is not necessarily required to use product-specific software. Therefore, this method can be used generally.

[0055] The proposed method provides the possibility of software control and automated solution even in an immersion objective lens to obtain motorized parfocality correction without performing automatic immersion or generally without applying immersion liquid. This method can no longer only achieve manually defined compensation but can also achieve a workflow that can run without manual intervention by the user. In other words, this method can also be executed by less trained users or even automatically without affecting the accuracy.

[0056] This also relates to, for example, a sample carrier or a reference carrier, which are no longer contaminated by the immersion medium when calibrating the immersion objective lens. Cleaning is omitted and reliable reusability is ensured. Since the use of immersion liquid is omitted, this method also saves significant time compared with the methods for performing parfocality correction to date. In addition, user acceptance can be improved, that is, the user may be more willing to perform the necessary offset correction, which can subsequently achieve a more correct imaging result. In particular, since the user finds the (previously) coordinated Z scale, collisions between the objective lens and the sample can also be avoided. However, slight manual correction may still be required when necessary. Extensive correction for a moving distance far outside the focal position that may cause the collision is no longer required.

[0057] According to different implementation variants, this method may include obtaining offset values for performing concentricity correction or concentric position correction by the following steps: obtaining the object position of the object with a reference objective lens without immersion, obtaining the object position of the object with an immersion objective lens without immersion, and obtaining the concentricity offset value of the concentricity offset between the immersion objective lens and the reference objective lens based on the object position obtained without immersion.

[0058] If not only concentricity correction but also parfocality correction is performed, the same object can be advantageously used as the object for determining the object position or for obtaining the focal position. This simplifies the method process and saves time because a large moving distance is not required to find another object. However, there is also the possibility of using different objects for the two corrections. This can achieve more accurate correction because in the case of separate objects, the correction can be optimized for the corresponding correction to be performed.

[0059] Optionally, a concentricity offset value can be determined based on the object position obtained without immersion liquid and at least one concentricity correction value. The concentricity correction value can at least approximately compensate for the missing immersion liquid and thus contribute to a concentricity correction with high precision.

[0060] For lateral correction (i.e., concentricity correction), it can be very approximately assumed that the difference in the lateral position (e.g., caused by the tilt of the optical element) between the determination with immersion liquid and the determination without immersion liquid is very small and can thus be set equal to zero. By not using the concentricity correction value or using a zero value for the concentricity correction value, the concentricity correction can be advantageously simplified and particularly more smoothly performed. Thus, the concentricity offset value between the objective lenses can be directly determined based on the obtained offset of the object structure in the image.

[0061] Advantageously, the corresponding target structure of the object is positioned at the image center by means of the sample stage, and thus the offset value to be corrected between these objective lenses can be directly determined by the travel distance of the sample stage required therefor. Advantageously, the sample stage is motorized for this purpose and can be controlled by the control unit. Alternatively, the concentricity offset value can be determined based on digital data or image data evaluating the offset effect and / or the scaling effect. A combination of both approaches is also possible.

[0062] Further optionally, the method can include compensating for the concentricity offset between the immersion objective lens and the reference objective lens by means of the obtained concentricity offset value. Compensation for the concentricity offset can be particularly achieved when changing between the reference objective lens and the immersion objective lens operating with immersion liquid or in this case.

[0063] As long as no differences are set forth below, the above-described embodiments for determining the concentricity offset value correspondingly apply to determining the parfocality offset value.

[0064] For performing concentricity correction, first the object positions of the object to be imaged are determined for the reference objective lens and the immersion objective lens, and the offset or misalignment of the immersion objective lens relative to the reference objective lens should be corrected.

[0065] The object positions of the object are determined for the two objective lenses without immersion liquid. In this context, "determining the object position" means: using the reference objective lens and the immersion objective lens to determine the positions of one or more features of the object in order to be able to determine the lateral displacement of the image based thereon. In this context, the main finding of the present inventors is that for an immersion objective lens, although it is designed to be used with an immersion liquid, the object position can be determined precisely enough even without immersion liquid to perform the concentricity correction according to the proposed method.

[0066] Based on the obtained object position, a concentricity offset value is determined by optionally considering a concentricity correction value, which represents the degree of concentricity offset between the immersion objective and the reference objective.

[0067] The concentricity correction value is used to correct the missing immersion liquid in the case of determining the object position using the immersion objective, and, if necessary, to correct the missing immersion liquid in the case of determining the object position using the reference objective (if the reference objective is also an immersion objective). In other words, the concentricity correction value can be regarded as an immersion correction value for correcting the concentricity offset. Thus, it is clear that the concentricity correction value between the objectives can be allocated to each objective, and it holds that: d ij = d i + d j . It is clear from the definition that the value d i of the objective in the absence of immersion liquid is equal to zero.

[0068] One or more concentricity correction values can be determined once in advance and stored in the proposed method for later application or, in other words, called from a storage unit within the scope of the proposed method. Additionally, there is the possibility, for example, in a computing unit, to parameterize the obtained concentricity correction values with respect to correlations with environmental parameters such as temperature, carrier substrate thickness, etc. and to derive the concentricity correction values for specific environmental conditions, as will be explained in more detail below.

[0069] "Determining the concentricity offset value based on the obtained object position" means that the obtained object position is considered directly or indirectly when determining the concentricity offset value. For example, the obtained object position can be corrected with the aid of the concentricity correction value, and then the corrected object position is used to determine the concentricity offset value.

[0070] Alternatively, the concentricity basic offset value of the basic concentricity offset between the immersion objective and the reference objective can first be determined based on the object position obtained in the absence of immersion liquid, and then this concentricity basic offset value can be corrected with the aid of the concentricity correction value. In this case, the concentricity offset value is determined based on the concentricity basic offset value and the concentricity correction value.

[0071] In a further optional method step, the concentricity offset between the immersion objective and the reference objective can then be compensated for with the aid of the obtained concentricity offset value. This compensation can be achieved by suitable measures, for example, moving or shifting the microscope stage in the x - direction and / or y - direction so that the object to be imaged is moved relative to the immersion objective. In digital image acquisition, the concentricity offset can also be compensated for digitally during the processing and presentation of the image data.

[0072] The obtained concentricity offset value can be stored for later use, such that the obtained concentricity offset value can be reused for a specific objective lens combination without having to re-obtain the object position. In other words, in the case of re-changing the objective lens, i.e., in the case of reusing a specific objective lens, the concentricity offset value obtained once can be directly used to compensate for the concentricity offset.

[0073] Concentricity correction simplifies the observation of the object to be imaged, because after completion of the concentricity correction, the object point located at the center of the image field is still at a substantially unchanged position at the center of the image after changing the objective lens, even if the magnification has changed. The concentricity correction can be performed together with the parfocality correction in order to correct the objective lens offset not only in the z-direction, but also in the x-direction and y-direction, and thus to improve the imaging quality. The advantages described with respect to the parfocality correction are correspondingly associated with the concentricity correction. In particular, the immersion liquid can be dispensed with, which is accompanied by a considerable time saving and a significantly reduced cleaning effort.

[0074] The imaging quality improved by the previous parfocality correction enables a more precise determination of the concentricity offset. Therefore, it may be advantageous to first perform the parfocality correction and then perform the concentricity correction.

[0075] According to a further embodiment variant, the method may include experimentally obtaining the parfocality correction value and / or the concentricity correction value using an immersion objective lens in the presence of an immersion liquid.

[0076] This means that, for example, the focal position of the immersion objective lens and / or the object position of the object can be determined once using the immersion objective lens in the presence of an immersion liquid. Based on this, the deviation of the focal position and / or the object position or the basic parfocality offset value and / or the basic concentricity offset value between using the immersion objective lens in the presence of an immersion liquid and without an immersion liquid can be obtained, and the parfocality correction value or the concentricity correction value can be determined accordingly.

[0077] The advantage of experimentally obtaining the parfocality correction value and / or the concentricity correction value is that all actual characteristics of the imaging optical system and the immersion objective lens, as well as possible unknown influencing factors, can be taken into account. Therefore, experimentally determining can provide very precise parfocality correction values and / or concentricity correction values.

[0078] According to a further embodiment variant, the method may have: computationally obtaining the parfocality correction value and / or the concentricity correction value by means of simulation and / or calculation.

[0079] This means that, for example, the focal position of the immersion objective and / or the object position of the object to be imaged can be determined by means of simulation and / or calculation. Based on this, it is possible to determine the deviation of the focal position and / or object position or the basic parfocality offset value and / or basic concentricity offset value between using the immersion objective with immersion and without immersion, and accordingly determine the parfocality correction value or concentricity correction value.

[0080] The advantage of computationally determining the parfocality correction value and / or concentricity correction value is that there is no need for time-consuming and resource-intensive experimental determination related to individual cases in the presence of immersion. This experimental determination is also associated with a high cleaning effort and can only be carried out by trained personnel. In contrast, the necessary computational determination can be carried out generally for specific device types or microscope types and / or, for example, (within the scope of remote maintenance) based on software if necessary. Thus, user interaction can possibly be omitted. In addition, the user acceptance for fully performing the offset correction can be increased because it is associated with little effort. Therefore, although the accuracy of the computationally determined parfocality correction value or concentricity correction value may be less than that of the experimentally determined parfocality correction value or concentricity correction value, in any case, such an offset correction can be achieved, which provides better imaging compared to imaging without any offset correction.

[0081] It is also possible to combine the experimental and computational determination of the parfocality correction value and / or concentricity correction value. The experimentally and / or computationally determined parfocality correction value or concentricity correction value can be stored, for example, in a storage unit, so that the correction value can be retrieved from the memory for later use without having to be determined again.

[0082] According to a further embodiment variant, the method can include: correcting the parfocality offset and / or concentricity offset compensated by means of the computationally determined parfocality correction value and / or concentricity correction value, determining the corrected parfocality correction value and / or corrected concentricity correction value based on the compensated parfocality offset and / or concentricity offset, and storing the corrected parfocality correction value and / or corrected concentricity correction value.

[0083] For example, the initial parfocality correction value and / or concentricity correction value can be rewritten using the corrected parfocality correction value or concentricity correction value, or the corrected parfocality correction value and / or corrected concentricity correction value can be stored in addition to the initial parfocality correction value or concentricity correction value. Subsequently, the stored corrected parfocality correction value and / or corrected concentricity correction value can be used for offset correction.

[0084] By combining the computational determination of the parfocality correction value and / or the concentricity correction value with experimental optimization, on the one hand, a very precise parfocality correction value or concentricity correction value can be obtained, enabling high-precision offset correction based thereon. On the other hand, the method can be executed in a time-saving manner because the computationally determined parfocality correction value or concentricity correction value is already close to the optimized parfocality correction value or concentricity correction value.

[0085] The corrected parfocality correction value or concentricity correction value can also be stored for later use.

[0086] According to a further embodiment variant, the parfocality correction value and / or the concentricity correction value can take into account influencing factors selected from the group consisting of: the composition of the immersion liquid, the temperature of the immersion liquid, the wavelength of the electromagnetic radiation used for imaging with the imaging optical system, the thickness of the carrier substrate, the refractive index of the carrier substrate, the distance of the object to the carrier substrate, the setting of the correction elements (such as correction rings or diaphragms) of the immersion objective, and the position of the object relative to the carrier substrate.

[0087] In other words, one of the mentioned influencing factors or boundary conditions or any combination of the mentioned influencing factors can be considered. In other words, the parfocality correction value or the concentricity correction value can be valid for a specific combination of influencing factors, for example, for a specific composition of the immersion liquid, and can be determined accordingly. The mutual influence between the influencing factors can also be considered.

[0088] In particular, the axial misalignment value can be affected by various influencing factors, and these influencing factors can be additionally considered to achieve even more precise offset correction. For example, temperature can affect the refractive index of the immersion liquid used.

[0089] The influencing factors can be considered by introducing them into the determination of the parfocality offset or the concentricity offset. In other words, the parfocality correction value or the concentricity correction value can together reflect such influencing factors.

[0090] The influence of one or more of these influencing factors on the parfocality correction value and / or the concentricity correction value can also generally be determined, stored, and applied to the specific parfocality correction value or concentricity correction value to be determined at a later time point. For example, a temperature correction factor can generally be determined and then considered when determining the specific parfocality correction value and / or concentricity correction value.

[0091] According to a further embodiment variant, the focal position can be determined automatically.

[0092] For this purpose, a hardware autofocus system (e.g., based on reflection, using propagation time measurement, and / or based on triangulation) or an image-based, computer-implemented autofocus method can be used. Advantageously, the automated determination of the focus position enables the automation of the proposed method, which can be accompanied by a reduction in the method duration and can eliminate user interaction.

[0093] According to a further embodiment variant, the object to be imaged can be an object permanently arranged on the optical system or arranged in a position-fixed manner on a carrier substrate.

[0094] The object can in particular have such a target structure that enables the determination of the focus position as precisely as possible. The advantage of an object permanently arranged at the optical system is that the optical system forms a stable reference as a stationary object and can thus be imaged under the same conditions at different points in time, enabling a convincing comparison between the images. Thus, when replacing another objective lens, the previously obtained images of the object or its target structure can continue to be used. Therefore, re-imaging the object with a reference objective lens can be dispensed with, and thus offset correction can be carried out smoothly and without a complex method flow.

[0095] Additionally or alternatively, a marker can be fixedly arranged on the carrier substrate as an object or target structure and / or the marker can be designed as an integral part of the carrier substrate. This can advantageously allow for the accurate and automated determination of parfocality and concentricity at different objective magnifications.

[0096] A further aspect of the invention relates to an assembly having an imaging optical system, the imaging optical system having an immersion objective lens and a reference objective lens and a device adapted to carry out the steps of one of the above-described methods.

[0097] Therefore, the above-described embodiments for explaining the method are also used to describe the proposed assembly. The advantages of the method are correspondingly associated with the assembly.

[0098] The device adapted to carry out these method steps can be designed, for example, as described below.

[0099] The device can include, for example, a device in the form of a processing and control unit, which is configured and designed to determine an offset value of the offset between the reference objective lens and the immersion objective lens by means of an image of the object.

[0100] Optionally, the device can include a device for compensating for the parfocality offset between the immersion objective lens and the reference objective lens, for example, in the form of a stage that can be moved in the z direction, possibly motorized, a corresponding movable focusing drive device, and / or one or more movable zoom lenses in the zoom body of the immersion objective lens.

[0101] In addition, the device may include a storage unit which is designed to store values such as, for example, the focal position of the objective lens in the absence of immersion liquid, the substantially parfocality offset value between the immersion objective lens and the reference objective lens in the absence of immersion liquid, the parfocality offset value between the immersion objective lens and the reference objective lens, the object position of the object, the substantially concentricity offset value between the immersion objective lens and the reference objective lens, and / or the concentricity offset value.

[0102] Optionally, the storage unit may be designed to store a corrected parfocality correction value and / or a corrected concentricity correction value.

[0103] The processing and control unit may, for example, be configured and designed to: determine the parfocality offset value between the immersion objective lens and the reference objective lens based on the stored focal position and / or the stored substantially parfocality offset value and the parfocality correction value, and generate and output a control signal which, by means of the determined parfocality offset value, causes compensation for the parfocality offset between the immersion objective lens and the reference objective lens. The processing and control unit may be implemented according to hardware and / or software and is physically designed as a single-piece or multi-piece unit.

[0104] In addition, the processing and control unit may be configured and designed to: determine the concentricity offset value between the immersion objective lens and the reference objective lens based on the stored object position and / or the stored substantially concentricity offset value and the concentricity correction value, and generate and output a control signal which, by means of the determined concentricity offset value, causes compensation for the concentricity offset between the immersion objective lens and the reference objective lens.

[0105] Further optionally, the processing and control unit may be configured and designed to: computationally determine the parfocality correction value and / or the concentricity correction value, and further optionally to determine a corrected parfocality correction value and / or a corrected concentricity correction value. For this purpose, the control unit may further optionally be configured and designed to take into account influencing factors selected from the group consisting of: the composition of the immersion liquid, the temperature of the immersion liquid, the wavelength of the electromagnetic radiation used to image with the imaging optical system, the thickness of the carrier substrate, the refractive index of the carrier substrate, the distance to the object to be imaged, the setting of the correction element of the immersion objective lens.

[0106] In addition, these devices may include means for automatically determining the focal positions of the immersion objective lens and the reference objective lens, or the processing and control unit may be correspondingly designed.

[0107] According to different implementation variants, the imaging optical system may be an optical microscope.

[0108] In an optical microscope, offset correction is particularly important for obtaining an image of sufficient quality.

[0109] A further aspect of the present invention relates to a computer program comprising instructions which cause the proposed components to implement one of the proposed methods.

[0110] Accordingly, the above-described embodiments for the method and the components are also used to describe the proposed computer program. The advantages of the method and the components are correspondingly associated with the computer program.

[0111] A computer program can be understood as program code that can be stored on a suitable medium and / or can be invoked via a suitable medium. For storing the program code, any medium suitable for storing software can be used, such as non-volatile memory installed in a controller, DVD, USB flash drive, flash memory card, etc. The invocation of the program code can be carried out, for example, via the Internet or an intranet or via other suitable wireless or wired networks.

[0112] The computer program can be stored on a non-volatile computer-readable medium and / or transmitted by means of a data carrier signal.

[0113] Further advantages of the present invention are apparent from the drawings and the associated description. The present invention will be explained in more detail below with reference to the drawings and the associated description. In the drawings:

[0114] Figure 1 A schematic diagram showing exemplary components;

[0115] Figure 2 A flowchart showing an exemplary method for offset correction of an immersion objective;

[0116] Figure 3 A flowchart showing another exemplary method for offset correction of an immersion objective;

[0117] Figure 4 A diagram showing for illustrating the basic parfocal offset;

[0118] Figure 5 A diagram showing for illustrating the parfocal offset;

[0119] Figure 6 Showing Figure 1 a block diagram of the exemplary components;

[0120] Figure 7a A diagram showing an imaging modality in which the sample is in direct contact with the immersion liquid;

[0121] Figure 7b A diagram showing an imaging modality in which the sample is behind the first substrate; and

[0122] Figure 7c A diagram showing an imaging modality in which the sample is positioned at a certain distance behind the carrier substrate.

[0123] It should be understood that other embodiments can be used and structural or logical changes can be made without departing from the scope of protection of the present invention.

[0124] When the expression “and / or” is used herein for several (two or more) elements, it means that each of the listed elements can be used alone or two or more of the listed elements can be used in any combination.

[0125] Figure 1 An embodiment of an assembly 200 having an imaging optical system 2 designed as an optical microscope is shown. The imaging optical system 2 includes a stage 6 on which a slide is arranged as a carrier substrate 5. The carrier substrate 5 is used to accommodate and position a sample to be observed by means of the stage 6. In addition, the imaging optical system 2 has an objective changer 7 into which an immersion objective 1 and a reference objective 3 are inserted, and the reference objective is an air objective. Of course, there can also be additional objectives, for example in the form of air objectives or immersion objectives. The objective changer 7 can be designed, for example, as an objective turret.

[0126] In addition, a microscope stand 8 is provided, which has a support 23 and a guide 9. The stage 6 can be moved along the guide in the z direction by means of a focusing drive device 10 in order to be able to focus the objectives 1, 3. In addition, the microscope stand 8 bears an eyepiece tube 12 on which an eyepiece 13 and a camera 11 are arranged.

[0127] In order to move the focusing drive device 10 along the guide 9 to focus the microscope, a motorized drive device can be provided as a focusing device 14, and the focusing device is connected to the processing and control unit 15 of the assembly 200 in a signal transmission manner, so that a control signal 16a can be transmitted from the processing and control unit 15 to the focusing device 14 to achieve focusing. Similarly, the stage 6 allows the carrier substrate 5 to be positioned preferably in a motorized manner and by means of a control signal 16b from the control unit 15.

[0128] The processing and control unit 15 is connected to a storage unit 17 in a signal transmission manner, so that values stored in the storage unit 17, such as parfocality correction values and / or concentricity correction values, can be called by the processing and control unit 15 and values generated by the processing and control unit 15 can be stored in the storage unit 17. The specific cooperation of the imaging optical system 2 with the processing and control unit 15 and the storage unit 17 will be described in more detail with reference to the remaining drawings below.

[0129] The manufacturing of the objective lenses 1, 3 and their installation in the objective lens changer 7 are related to tolerances, which may cause deviations in the foci and / or centers of the objective lenses 1, 3. The deviations lead to a deterioration of the imaging quality or to laborious readjustment when changing the objective lens. As a secondary effect, the user frequently searches for the focus, which may cause collisions in the case of a small working distance of the immersion objective lens, and then the collisions may cause damage to the objective lens or the microscope module and associated high repair costs. In order to largely prevent this, offset correction is carried out with the aid of offset values to compensate for the tolerances, the accuracy of which is higher than the accuracy of the tolerance chain.

[0130] In this embodiment, the offset values are determined based on the imaging of the object 4 and the target structure fixedly arranged on the stage 6 according to the position, as will be explained in more detail below. Alternatively, the object 4 can also be arranged on or integrated into the carrier substrate 5. The target structure of the object 4 is designed such that the focus position can be determined when imaging the object 4 with one of the objective lenses 1, 3.

[0131] Figure 2 Schematically shows the flow of an exemplary method 100 for offset correction of an immersion objective lens 1 (for example, the immersion objective lens 1 of the imaging optical system 2 of the components shown in Figure 1 .

[0132] In step 101, the object 4 or its target structure is imaged without immersion fluid using a reference objective lens and an immersion objective lens respectively.

[0133] In the subsequent step 102, based on the imaging of the object 4, the basic offset values of the offset between the reference objective lens 3 and the immersion objective lens 1 are determined without immersion fluid, and these basic offset values reflect the offset between the two objective lenses 1, 3 without immersion fluid. Optionally, a plurality of basic offset values can be determined, such as the basic offset values of parfocality offset and / or concentricity offset.

[0134] In step 103, based on these basic offset values, the offset values between the reference objective lens 3 and the immersion objective lens 1 are determined with immersion fluid, and these offset values are stored in step 104.

[0135] When changing the objective lens, for example, after switching from the reference objective lens 3 to the immersion objective lens 1 using the objective lens changer 7, in step 105, the determined offset values are used to perform offset correction in order to compensate for the offset of the objective lenses 1, 3 and to achieve high-quality imaging.

[0136] Figure 3 Schematically shows another exemplary method 100 for offset correction. According to Figure 3 's method 100 provides parfocality correction and concentricity correction, as will be explained in more detail below.

[0137] Method 100 begins in step 110 with imaging of object 4 by means of reference objective 3 without immersion liquid. Object 4 can in particular be an object 4 which is arranged stationary on the microscope and has a target structure (for example a line pattern by means of which automated focusing can be carried out). In particular, an image stack can be generated in the z direction. "Stationary" can mean here, for example, that the target structure is brought to a position defined relative to the optical system by means of a controllable stage 6 and a z drive.

[0138] In step 111, the focal position z1 of reference objective 3 is determined based on the imaging of object 4 or the associated image stack. For this purpose, the structural features of the target structure of object 4 are used and its contrast and / or the resolution of the image are evaluated. Based on the images generated at different distances between reference objective 3 and the target structure in the z direction (i.e., the image stack in the z direction), the distance at which sharp imaging is produced can be determined. The associated position of reference objective 3 is its focal position z1. The determination of the focal position z1 can preferably be carried out automatically by means of a so-called autofocus function. The determined focal position z1 is subsequently used to determine the parfocality offset value.

[0139] In step 112, the imaging of object 4 is used to determine the object position by means of reference objective 3 without immersion liquid. For this purpose, the same imaging as in step 111 or the imaging of the z image stack can be used. Preferably, the imaging corresponding to the focal position z1 according to step 111 can be used. Here, using the focused imaging can enable a more precise determination of the object position. However, in principle, the object position can also be determined based on other imaging of object 4 or separate imaging of object 4 or other objects 4 can be used. Therefore, method steps 111 and 112 can be carried out in any order and / or overlapping in time. The determined object position is subsequently used to determine the concentricity offset value.

[0140] To avoid a changeover process in the automated process, it is advantageous to first determine the focus z1 of reference objective 3 on the target structure and position the target structure in the center of the image field. Then a changeover to immersion objective 1 is carried out in order to also determine z2 and the object position there (see the following description of steps 121 and 122). The focus value and the position value of the additional objective are determined without having to return to the positioning of reference objective 3 because its values have already been determined.

[0141] Method 100 proceeds from step 111 or step 112 to step 120. In step 120, after changing from reference objective 3 to immersion objective 1, step 110 is carried out in a similar manner with immersion objective 1 instead of reference objective 3, i.e., imaging of object 4 is carried out by means of immersion objective 1 without immersion liquid, whereupon an image stack can in turn be generated in the z direction.

[0142] Optionally, before step 120, a rough or approximate first offset correction (intermediate step 150) can be performed on the immersion objective 3. Such an approximate offset correction can include parfocality correction and / or concentricity correction, and is performed, for example, based on previously determined offset values, such as the parfocality basic offset value (e.g., for correcting the missing immersion liquid). Thereby, subsequently, finding the focus position and / or the object position can be made easier and thus automation can be achieved more robustly.

[0143] In step 121, similar to determining the focus position z1 of the reference objective 3, the focus position z2 of the immersion objective 1 is determined without immersion liquid. The determination of the focus position z2 is preferably also carried out automatically with the aid of a so-called autofocus function.

[0144] In step 130, then, the processing and control unit 15 determines the parfocality basic offset V 1-2 of the parfocality basic offset value, which indicates the parfocality offset between these two objectives 1, 3 without immersion liquid.

[0145] This situation is schematically shown in Figure 4 . In Figure 4 , the reference objective 3 (hereinafter also referred to as objective 1) is shown on the left and the immersion objective 1 (hereinafter also referred to as objective 2) is shown on the right. For the two objectives 1, 3, their focus positions z1, z2 have been determined respectively in such a way that an image stack of the object 4 arranged on the slide 5 is recorded by means of the image sensor 18 of the autofocus system in the z direction and then evaluated. That is to say, the focus positions z1, z2 are determined automatically. The position difference in the z direction of these two objectives 1, 3 required to obtain a correspondingly focused imaging is the parfocality basic offset V 1-2 between the reference objective 3 (i.e., objective 1) and the immersion objective 1 (i.e., objective 2), that is, the parfocality offset without immersion liquid.

[0146] In other words, Figure 4 shows the parfocality basic offset V 1-2 between the two objectives, namely the reference objective 3 and the immersion objective 1, the value of which is obtained by calibration without immersion liquid. For this purpose, the focus positions z1, z2 are determined for each of the objectives 1, 3 at which the best imaging of the object 4, more precisely the target structure of the object 4, on the sensor is achieved. Here, the immersion objective is also used without immersion liquid in order to simplify the operation and avoid manual interaction by the user. The parfocality basic offset V is obtained from the difference in the focus positions z1, z2. 1-2= z2 - z1, which indicates which offset needs to be corrected in order to change from objective lens 1 to objective lens 2 without immersion liquid and thereby image the sample onto the image sensor 18 with optimal resolution.

[0147] Refer again to Figure 3 , in step 131, based on the obtained focal positions z1, z2 in the form of the parfocality basic offset value and the parfocality correction value c 1-2 , the parfocality offset V~ 1-2 between the immersion objective lens 1 and the reference objective lens 3 is obtained. In the intermediate step 132, for this purpose, the parfocality correction value c 1-2 is called from the storage unit 17.

[0148] Figure 5 shows the case where there is a parfocality offset V~ 1-2 between the immersion objective lens 1 and the reference objective lens 3 in the presence of immersion liquid, that is, the immersion objective lens 3 is used together with the immersion liquid 19 (which is a water-glycerol mixture in this embodiment). The object 4 to be imaged is located behind the cover glass 21 here. For other imaging modalities, refer to Figures 7a to 7c and its description.

[0149] In other words, Figure 5 shows which parfocality offset V~ 1-2 must be considered when changing between the objective lenses 1 and 3 in order to correct the misalignment in the observation case where the immersion objective lens 3 operates with immersion liquid. In the simple approximation shown here, the parfocality correction value c 1-2 corresponds to the difference between the parfocality basic offset value of the parfocality basic offset V 1-2 and the parfocality offset value of the parfocality offset V~ 1-2 , that is, it holds that: c 1-2 = V~ 1-2 - V 1-2 .

[0150] The parfocality correction value c 1-2 can be obtained experimentally or theoretically, for example, by experimental calibration or by optical simulation. In other words, the parfocality correction value c 1-2 (also see Figure 4 ) can be obtained experimentally from the measured offsets of the focal positions z1, z2 of different configurations (such as according to the position K of the correction ring, the temperature T, or the thickness D of the carrier substrate 5), however, it can also be obtained from theoretical optical calculations or from a combination of these schemes.

[0151] Refer again to Figure 3, in step 133, the parfocality offset V~ between the immersion objective lens 1 and the reference objective lens 3 1-2 The parfocality offset value is stored, for example, in the storage unit 17.

[0152] The stored parfocality offset value can then be recalled and used for parfocality correction when needed. Thus, if, for example, when applying the microscope, the reference objective lens 3 is replaced with the immersion objective lens 1 and the immersion objective lens 3 is used with immersion liquid, then in step 134, the parfocality offset V~ between the immersion objective lens 1 and the reference objective lens 3 is compensated for by means of the obtained parfocality offset value. i-j .

[0153] Steps 110, 111, 120, 121 and 130 to 134 of method 100 are used for parfocality correction. In this embodiment, additionally, centricity correction is performed according to steps 110, 112, 120, 122 and 140 to 144. However, centricity correction can also be omitted or only centricity correction can be performed. However, since centricity correction is performed more precisely with the aid of the focused image, it is advisable to previously determine and set the focus position.

[0154] For centricity correction, as already mentioned, in step 112, first the object position of the object 4 to be imaged is determined with the reference objective lens 3 without immersion liquid. After imaging the object with the immersion objective lens without immersion liquid in step 120, in step 122, the object position of the object 4 to be imaged is determined with the immersion objective lens 3.

[0155] In step 140, the centricity basic offset value is then determined by the processing and control unit 15 according to the obtained object positions, which indicates the centricity offset between the two objective lenses 1 and 3 without immersion liquid.

[0156] In step 141, the centricity offset value of the centricity offset between the immersion objective lens 1 and the reference objective lens 3 is then determined based on the centricity basic offset value. For example, the centricity offset value can correspond to the centricity basic offset value. Optionally, a centricity correction value can be considered when determining the centricity offset value, and this centricity correction value can be recalled from the storage unit 17 in intermediate step 142.

[0157] In step 143, the centricity offset value is stored, for example, in the storage unit 17.

[0158] The stored centricity offset value can then be recalled and used for centricity correction when needed. Thus, if, for example, when applying the microscope, the reference objective lens 3 is replaced with the immersion objective lens 1 and the immersion objective lens 3 is used with immersion liquid, then in step 144, the centricity offset between the immersion objective lens 1 and the reference objective lens 3 is compensated for by means of the obtained centricity offset value.

[0159] As indicated by the dotted line in the middle Figure 3 shown, method 100 can correspondingly be continued to be used for additional objective lenses, especially for additional immersion objective lenses. Figure 6 Shown for the purpose of illustrating the Figure 1 components 200 corresponding to the Figure 2 or Figure 3 working mode of method 100 is a block diagram.

[0160] As already mentioned, component 200 includes a reference objective lens 3 (here called objective lens No. 1), which has a focal position z1 without immersion. In addition, there is an immersion objective lens 3 (here called objective lens No. 2), which has a focal position z2 without immersion. Optionally, there may be additional objective lenses with subordinate focal positions z, as indicated in Figure 5 . For observing the object 4 to be imaged, it is possible to switch between, for example, a reference objective lens 3 with a low magnification and an immersion objective lens 1 with a higher magnification and good resolution due to immersion. Here, it is necessary to perform offset correction, especially parfocality correction and optionally additionally perform concentricity correction in order to achieve good imaging quality without refocusing.

[0161] To perform the parfocality correction of method 100, the focal positions z1, z2 of the reference objective lens 3 and the immersion objective lens 1 are determined without immersion, and thus the parfocality basic offset V 1-2 of the parfocality basic offset value is obtained. Here, V 1-2 = z2 - z1 holds. If the parfocality basic offset of the object position is determined in a series of objective lenses, the parfocality basic offset is simply obtained by adding up the respective basic offset values.

[0162] V 1-i = V 1-2 + V 2-3 + … + V i-1-i = (z2 - z 1) + (z3 - z 2) + … + (z i - z i-1 ) = z i

[0163] - z1. Thus, it is clear that the parfocality basic offset value between two objective lenses only relates to the determined focal positions of these two objective lenses and, very generally, for the performed measurement sequence, it holds that:

[0164] V i-j = z j - z i .

[0165] Homofocal basic offset V i-j The obtained homofocal basic offset value is stored in the storage unit 17 of the component 200 for later use.

[0166] For example, in order to obtain the homofocal offset V~ Figure 3 in step 131 of method 100, first the homofocal correction value c 1-2 is obtained. This can be done experimentally or based on calculations (e.g., based on the optical design on which the imaging optical system is based) and, for example, with the aid of the processing and control unit 15. When obtaining the homofocal correction value c 1-2 , additional influencing factors 20 can be taken into account, and their influence on homofocality can be obtained by means of additional measurements or calculations. The homofocal correction value c 1-2 can be stored in the storage unit 17 and recalled from this storage unit at a later point in time (e.g., in step 133 of method 100 according to 1-2 ). Parameterization can be carried out here for the structure of the object 4 or the variable optical settings. This involves, for example, performing calibration with different correction ring settings, but also taking into account the correlations with temperature T, the thickness D of the carrier substrate, the sample position relative to the substrate, or using an immersion liquid 19 with a variable refractive index, such as a water-glycerol mixture. Figure 3 If the objective lens has been replaced, the processing and control unit 15 obtains the homofocal offset V~

[0167] based on the homofocal basic offset value and the homofocal correction value c 1-2 of the homofocal offset value. In this case, for the focal position f2 of the immersion objective lens in the presence of an immersion liquid, it holds that: f2 = f1 + V 1-2 + c 1-2 , where f1 is the current focal position of the reference objective lens 3, which corresponds to the focal position z1 or can also be different from it. If the influencing factor 20 is taken into account, the focal position f2 can generally be obtained according to f2 = f1 + f(V 1-2 ; c 1-2 ; K; T; D;...). 1-2

[0168] In other words, stored in the storage unit 17 is the homofocal basic offset value of the homofocal basic offset V i-j obtained during calibration without an immersion liquid between objective lenses i and j. When replacing the objective lens, the homofocal basic offset V i-j obtained without an immersion liquid is corrected by the processing and control unit 15 to obtain the correct homofocal offset V~ i-jThe parfocality offset value. The correction is dominated in particular by the imaging effect of the immersion liquid, but optionally additional influencing factors 20 can be taken into account.

[0169] To compensate for the parfocality offset V~ 1-2 , for example according to Figure 3 step 134 of method 100, the processing and control unit 15 generates a corresponding control signal 16, which is output to the focusing device 14 to cause a change in the distance between the object 4 and the immersion objective 1 in the z-direction.

[0170] As already mentioned, the concentricity correction is carried out similarly.

[0171] The proposed technical solution enables the following workflow, which performs the parfocality correction fully automatically and in particular even in the case of an immersion objective without the required immersion liquid.

[0172] In Figures 7a to 7c different imaging morphologies of the immersion objective 3 are shown. Here, the left side of the drawing shows the case without immersion liquid respectively, and the right side shows the case with immersion liquid. In addition, the corresponding parfocality offset V I , which occurs between the operation of the immersion objective 1 without immersion liquid and with immersion liquid and is taken into account in the proposed method by means of the parfocality correction value c i-j considered.

[0173] In Figure 7a the object 4 to be imaged is arranged on the slide 5 and is in direct contact with the immersion liquid 19 during immersion. In addition, an embedding medium 24 is arranged on the side of the carrier substrate 5 facing away from the image sensor 18.

[0174] In Figure 7b the object 4 to be imaged is arranged behind a first substrate, for example a cover glass 21. In this case, the immersion liquid 19 is located between the cover glass 21 and the immersion objective 1.

[0175] In Figure 7c the object 4 to be imaged is arranged at a distance behind the slide 5. In this case, the immersion liquid 19 is located between the slide 5 and the immersion objective 1.

[0176] Because in different imaging morphologies the light beam 22 travels different lengths of path in the immersion liquid and causes different medium transitions, slightly different parfocality offsets V I caused by the immersion are generated. Therefore, in order to perform the parfocality correction as precisely as possible, it is recommended to determine and use a separate parfocality correction value c i-j for each imaging morphology. The same can be done when performing the concentricity correction.

[0177] List of reference signs

[0178] 1 Immersion objective

[0179] 2 Imaging optical system

[0180] 3 Reference objective

[0181] 4 Object

[0182] 5 Carrier substrate

[0183] 6 Stage

[0184] 7 Objective changer

[0185] 8 Microscope stand

[0186] 9 Guide

[0187] 10 Focus drive

[0188] 11 Camera

[0189] 12 Eyepiece tube

[0190] 13 Eyepiece

[0191] 14 Focusing device

[0192] 15 Processing and control unit

[0193] 16a, 16b Control signals

[0194] 17 Storage unit

[0195] 18 Image sensor

[0196] 19 Immersion liquid

[0197] 20 Influence factor

[0198] 21 Cover glass

[0199] 22 Light beam

[0200] 23 Support

[0201] 24 Embedding medium

[0202] 100 Method

[0203] 101 Imaging the object with the reference objective and the immersion objective without immersion liquid

[0205] 102 Determining the basic offset value of the offset between the reference objective and the immersion objective from the imaging of the object

[0207] 103 Obtain the offset value for the offset between the reference objective lens and the immersion objective lens

[0209] 104 Store the offset value

[0210] 105 Perform offset correction using the obtained offset value

[0211] 110 Image the object using the reference objective lens without immersion liquid

[0213] 111 Obtain the focal position of the reference objective lens without immersion liquid

[0215] 112 Obtain the object position of the object using the reference objective lens without immersion liquid

[0217] 120 Image the object using the immersion objective lens without immersion liquid

[0219] 121 Obtain the focal position of the immersion objective lens without immersion liquid

[0221] 122 Obtain the object position of the object to be imaged using the immersion objective lens without immersion liquid

[0223] 130 Obtain the parfocality basic offset value of the parfocality offset between the immersion objective lens and the reference objective lens according to the focal positions obtained without immersion liquid

[0225] 131 Obtain the parfocality offset value of the parfocality offset between the immersion objective lens and the reference objective lens based on the parfocality basic offset value and the parfocality correction value

[0227] 132 Call the parfocality correction value

[0228] 133 Store the parfocality offset value

[0229] 134 Compensate for the parfocality offset between the immersion objective lens and the reference objective lens

[0230] 140 Obtain the concentricity basic offset value of the concentricity offset between the immersion objective lens and the reference objective lens according to the object positions obtained without immersion liquid

[0232] 141 Obtain the concentricity offset value of the concentricity offset between the immersion objective lens and the reference objective lens based on the concentricity basic offset value

[0234] 142 Call the concentricity correction value

[0235] 143 Store the concentricity offset value

[0236] 144 Compensate for the concentricity offset between the immersion objective lens and the reference objective lens

[0237] 150 Perform an approximate offset correction

[0238] 200 Component

[0239] c i-j The parfocality correction value for correcting the basic parfocality offset between objective lenses i and j

[0241] d The concentricity correction value for correcting the basic concentricity offset

[0242] f1 The focal position of the reference objective lens

[0243] f2 The focal position of the immersion objective lens (in the presence of immersion liquid)

[0244] D The thickness of the slide

[0245] K The position of the correction ring

[0246] T Temperature

[0247] V i-j The basic parfocality offset between objective lenses i and j

[0248] V~ i-j The parfocality offset between objective lenses i and j

[0249] V I The parfocality offset caused by the immersion liquid

[0250] z1 The focal position of the reference objective lens without immersion liquid

[0251] z2 The focal position of the immersion objective lens without immersion liquid

Claims

1. A method (100) for determining an offset value for performing offset correction on an immersion objective (1) of an imaging optical system (2), the method (100) comprising the following steps: - imaging (101, 110, 120) an object (4) using a reference objective (3) of the optical system (2) and the immersion objective (1) without immersion liquid, and - determining (103, 131, 141) an offset value of the offset between the reference objective (3) and the immersion objective (1) by means of an imaging of the object (4) in the presence of an immersion liquid.

2. The method (100) according to claim 1, comprising: - determining (102, 130, 140) a basic offset value of the offset between the reference objective (3) and the immersion objective (1) by means of an imaging of the object (4), The offset value is obtained based on the basic offset value. 3 . The method according to claim 1 , wherein an offset value is ascertained for carrying out a parfocality correction and / or a concentricity correction.

4. The method (100) according to claim 3, wherein determining the offset value for performing the parfocality correction comprises the following steps: - determining (111) the focal position (z1) of the reference objective (3) of the imaging optical system (2) without immersion liquid, - determining (121) the focal position (z2) of the immersion objective (1) without immersion liquid, and Based on the determined focus position (z1, z2) and at least one parfocality correction value (c 1-2 ), calculate (131) the parfocality offset (V~) between the immersion objective lens (1) and the reference objective lens (3) 1-2 )’s parfocality offset value.

5. The method (100) according to claim 4, comprising: - determining (130) a basic deviation (V) of the parfocality between the immersion objective (1) and the reference objective (3) based on the focal position (z1, z2) determined without immersion liquid 1-2 )'s parfocality basic offset value, wherein based on the parfocality basic offset value and the parfocality correction value (c 1-2 ) to obtain the parfocality offset value.

6. The method (100) according to any one of claims 3 to 5, wherein determining the offset value for performing the concentricity correction comprises the following steps: - determining (112) the object position of the object (4) by means of the reference objective (3) without immersion liquid, - determining (122) the object position of the object (4) by means of the immersion objective (1) without immersion liquid, and - determining (141) a concentricity offset value of the concentricity offset between the immersion objective (1) and the reference objective (3) based on the object position determined without immersion liquid.

7. The method (100) according to claim 6, wherein the isocentricity offset value is determined based on an object position determined without immersion liquid and at least one isocentricity correction value (d).

8. The method (100) according to any one of claims 4 to 7, comprising: - experimentally determining the parfocality correction value (c) using the immersion objective (1) in the presence of immersion liquid 1-2 ) and / or the concentricity correction value (d).

9. The method (100) according to any one of claims 4 to 8, comprising: - Calculate the parfocality correction value (c 1-2 ) and / or the concentricity correction value (d).

10. The method (100) according to claim 9, comprising: - by means of the calculated parfocality correction value (c 1-2 ) and / or the parfocality offset compensated by the concentricity correction value d (V~ 1-2 ) and / or concentricity deviation, -Based on the compensated parfocality shift (V~ 1-2 ) and / or concentricity offset to obtain a corrected parfocality correction value and / or a corrected concentricity correction value, and - Storing the corrected parfocality correction value and / or the corrected concentricity correction value.

11. The method (100) according to any one of claims 4 to 10, wherein the parfocality correction value (c 1-2 ) and / or the concentricity correction value (d) taking into account an influencing factor (20) selected from the group consisting of: The composition of the immersion liquid, the temperature (T) of the immersion liquid, the wavelength of the electromagnetic radiation used for imaging with the imaging optical system (2), the thickness (D) of the carrier substrate (5), the refractive index of the carrier substrate (5), the distance of the object (4) from the carrier substrate (5), the setting of the correction element of the immersion objective (1) and the position of the object (4) relative to the carrier substrate (5). 12 . The method ( 100 ) as claimed in claim 4 , wherein the focal position ( z1 , z2 ) is determined automatically.

13. The method (100) according to any one of the preceding claims, wherein the object (4) is an object which is permanently arranged on the optical system (2) or is arranged in a fixed position on a carrier substrate (5).

14. An assembly (200) comprising: - an imaging optical system (2) having an immersion objective (1) and a reference objective (3), and - means adapted to carry out the steps of the method (100) according to any one of claims 1 to 13.

15. The method (100) according to any one of claims 1 to 13 or the assembly (200) according to claim 14, wherein the imaging optical system (2) is an optical microscope.

16. A computer program comprising instructions for causing a component (200) according to claim 14 or 15 to implement a method (100) according to any one of claims 1 to 13 or 15.

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