Method for correcting a spherochromatic aberration and microscope

Abstract

The present invention relates to a method for correcting a spherochromatic aberration that occurs when examining a sample with a microscope. The microscope comprises an objective lens and a corrective device arranged in a beam path of the microscope for correcting a spherical aberration. According to the method, data for describing the spherical aberration as a function of a wavelength of light imaged by the microscope are provided. A sample is examined with the microscope, and at least one spectral range of the light imaged by the microscope is determined.A control parameter of the correction device is determined based on the provided data describing the spherical aberration as a function of wavelength for the at least one determined spectral range, in order to minimize the spherical aberration for the at least one determined spectral range. The correction device is then set to the determined control parameter. The invention further relates to a microscope, which is in particular a light microscope.

Description

[0001] The present invention relates firstly to a method for correcting a spherochromatic aberration that occurs when examining a sample with a microscope. The microscope comprises an objective lens and a corrective device arranged in a beam path of the microscope for correcting a spherical aberration. Furthermore, the invention relates to a microscope, which is in particular a light microscope.

[0002] A light microscope produces high-resolution images of sample structures. With samples that are at least partially transparent, it is also possible to look inside the sample. This is typical for medical and biological samples. The optical image quality depends primarily on elements and scales located in the optical path, which are considered imaging parameters. Most of these parameters, such as the shape, thickness, and spacing of lenses, are well-defined and controlled by the microscope manufacturer. Other parameters vary depending on the specific microscopic examination of a particular sample; these include the properties of the sample itself, the thickness of any coverslip used, and the temperature of the sample and the surrounding chamber. These parameters are therefore outside the control of the microscope manufacturer.For these parameters, which can also be referred to as sample space parameters, the actual imaging situation for the specific microscopic examination of the respective sample can deviate from the ideal situation assumed in the optical design, thus leading to aberrations. These deviations, as long as they occur homogeneously across the irradiated area, result in spherical aberrations. A proven method in the prior art for compensating these influences, which are very significant, at least for high-aperture objectives, is a mechanical correction ring on or in the objective, through which lenses within the objective can be shifted in the axial direction, a process that can also be automated. In principle, other adaptive optics can also be used for compensation.

[0003] JP 4554174 B2 discloses a solution for correcting optical aberrations caused by the thickness of a coverslip and a transparent sample holder. The correction should also be possible when the focus position of an objective lens relative to the sample is changed.

[0004] US Patent 9,741,122 B2 discloses a method for determining the distance between two optical interfaces spaced apart along a first direction. A pattern is projected onto a plane perpendicular to the first direction. An image of the pattern projected onto the plane is taken. These steps are repeated for various positions in the first direction, the different positions covering a region in the first direction in which the two optical interfaces lie. A first image is determined from these images, where the plane onto which the pattern captured with the first image was projected coincides with one of the two optical interfaces or has the smallest distance along the first direction to the first optical interface. The position of the first image in the first direction is determined. A second image is also determined in the same manner, and its distance from the first image is calculated.

[0005] From US 9,411,142 B2, a microscope system is known which includes an objective and a correction collar configured to move a lens within the objective and correct spherical aberration. A control unit of the microscope system is configured to receive multiple inputs from an observer before observation of a sample, specifying relative positions of the objective with respect to the sample. Multiple rotation angles of the correction collar are set, and an imaging device is caused to acquire multiple image data segments of the sample, each corresponding to a rotation angle of the correction collar. A contrast evaluation method calculates a score from each of the acquired image data segments, indicating the contrast of each image.

[0006] EP 3 118 665 B1 describes a microscopy system comprising a microscope device with an objective and a correction unit for correcting spherical aberration. An associated refractive index calculation unit is configured to calculate the refractive index of a sample at a target position within the sample based on a multitude of target values. Each target value corresponds to a degree of spherical aberration that occurs in the microscope device when an observation target plane is located at a different position within the sample along an optical axis.

[0007] WO 2009 / 096522 A1 discloses an aberration correction control device for a microscope. A control unit measures the temperature of oil, which is arranged as an oil immersion between a lens and a coverslip. The control unit determines the necessary rotation angle of a correction ring for aberration correction, based on a combination of the current oil temperature and the thickness of the coverslip, using a correction value table. A motor is then controlled to adjust the calculated rotation angle of the correction ring.

[0008] From US Patent 6,563,634 B2, a microscope is known with an aberration correction objective facing a sample and featuring an aberration correction lens that corrects aberrations caused by errors in the thickness of a cover, Petri dish, or slide. A motion detector is used to detect the movement of the aberration correction lens. A focusing unit is used to move the sample. A driver unit drives the focusing unit. A processing unit determines a measure of defocusing based on the movement detected by the motion detector. Based on this, the driver unit drives the focusing unit to focus the sample with the objective.

[0009] EP 3 112 919 A1 discloses an optical device comprising an optical module with an optical element and a control module for controlling the optical device. The optical module includes a subsystem with a memory and an actuator and / or a sensor. The memory contains instructions for setting the actuator and / or sensor of the subsystem. The control module is designed such that, during operation of the optical device, it reads the instructions from the memory of the subsystem and controls the actuator and / or sensor of the subsystem according to the instructions.

[0010] German patent DE 10 2018 126 000 A1 discloses a microscope objective that includes an adjustable correction mechanism for correcting an aberration. The objective incorporates a non-volatile memory chip on which a control parameter, specific to the aberration to be corrected and unique to the objective, is stored. The correction mechanism can be adjusted using this control parameter.

[0011] German patent DE 10 2018 126 021 B3 discloses a method for correcting an imaging aberration in a microscope system comprising a microscope and an optical component. A corrective agent contained in the optical component is adjusted to correct the imaging aberration. A control variable, specific to the optical component and associated with the imaging aberration, for adjusting the corrective agent is received from a remote storage module via a data transmission network.

[0012] From DE 10 2018 126 007 A1, a method for correcting a spherical aberration in a microscope is known. The microscope comprises an objective lens and a coverslip or support glass. A corrective element arranged in the objective lens serves to correct the spherical aberration. The microscope determines the refractive index of an optical medium bordering the coverslip or support glass and / or the thickness of the coverslip or support glass along the optical axis of the objective lens in order to correct the spherical aberration.

[0013] One cause of spherical aberrations is that microscopes use light of different wavelengths to examine samples. In principle, a microscope's spherical aberrations can only be minimized for light of a single wavelength, meaning that even in an ideal design, it cannot be equally well-suited for all wavelengths. Therefore, the design typically focuses primarily on the focal chromatic aberration (known as longitudinal chromatic aberration) and the lateral chromatic aberration (known as transverse chromatic aberration). However, all optical aberrations, such as all Zernike parameters, are chromatically dependent. The chromatic dependence of spherical aberration leads to spherochromatism. Furthermore, all of the sample space parameters mentioned above can exhibit a chromatic influence.

[0014] Starting from the prior art, the object of the present invention is to minimize spherochromatism as an imaging error when microscopy a sample with a microscope.

[0015] The aforementioned problem is solved by a method according to the attached claim 1 and by a microscope according to the attached dependent claim 12.

[0016] The method according to the invention serves to correct a spherochromatic aberration that occurs when examining a sample with a microscope. The microscope is a light microscope that images the sample using light reflected from the sample, light emitted by the sample, and / or light transmitted through the sample, where the term "light" here includes not only visible light but also ultraviolet and infrared radiation. The spherochromatic aberration is a spherical aberration that depends on the wavelength of the light imaged by the microscope.

[0017] The microscope comprises an objective lens and a correction device arranged in a beam path of the microscope for correcting a spherical aberration. The correction device is preferably arranged in the objective lens. The correction device preferably comprises at least one lens and an actuator for moving the lens along an optical axis of the objective lens. Alternatively, and preferably, the correction device is designed as a correction ring that is manually operated.

[0018] In one step of the process, data is provided that describes the spherical aberration as a function of a wavelength of the light imaged by the microscope. This preferably occurs before the microscope is used for its intended purpose, in which a sample is examined. The data describes properties of the objective lens and preferably also properties of other components involved in imaging the sample, such as a support glass, a cover glass, and / or an immersion medium located above the cover glass or support glass. This data can be generated from data provided by the objective lens or microscope manufacturer and / or determined through measurement series. The data is preferably stored in a memory.

[0019] When examining a sample with a microscope, a spectrum is determined in the form of at least one spectral range of the light imaging the sample. For example, the wavelength that is characteristic of and represents this at least one spectral range can be determined. Preferably, only one wavelength is determined if the imaging light consists essentially of only a single spectral range or if only a single spectral range is to be considered for microscopy of the sample.

[0020] In a further step, a setpoint for the correction device is determined based on the provided data describing the spherical aberration as a function of wavelength, specifically for the at least one determined spectral range, with the aim of minimizing the spherical aberration for this at least one determined spectral range. For this purpose, the correction device must be set to the determined setpoint, and the sample is then examined under the microscope. Setting the correction device to the determined setpoint can be automated if the correction device includes an actuator, or it can be done manually if the correction device consists of a correction ring. For manual setting, the determined setpoint is preferably displayed so that an operator can adjust the correction device accordingly.The minimization of the spherical imaging error for at least one determined spectral range should lead to the minimization or correction of the spherochromatic imaging error, which would occur when microscopy the sample with the light that precisely images this sample if the correction medium were to remain in its reference position.

[0021] A particular advantage of the method according to the invention is that the optical performance of the microscope is significantly improved by correcting the spherochromatic aberration.

[0022] In many applications, the at least one spectral range to be determined does not need to be measured but arises directly from the application itself. Therefore, the at least one spectral range is preferably determined from the spectrum of an excitation beam. The excitation beam is directed onto the sample for microscopy. Alternatively, the at least one spectral range is preferably determined from the spectrum of emission radiation shifted by a Stokes shift when the excitation beam is directed onto the fluorescent sample for microscopy, thereby inducing a Stokes shift in the excitation beam. Alternatively, the at least one spectral range is preferably determined from the spectrum of a color filter used for microscopy. This is the spectral range that the color filter allows to pass through.The at least one spectral range is preferably determined from a spectrum of a dye used for microscopy. This is the spectral range corresponding to the dye's color. Alternatively, and preferably, the at least one spectral range is determined from a measured spectrum of the imaging light.

[0023] In preferred embodiments, the at least one spectral range of the light imaging the sample by the microscope is determined by identifying a wavelength characteristic of that spectral range. The at least one spectral range is thus represented by its characteristic wavelength, allowing the provided data for describing the spherical aberration as a function of wavelength to be applied to it. This determination is preferably achieved by calculating the spectral data obtained from the application, as mentioned above, or from the measured spectrum of the imaging light. These embodiments are particularly suitable when the light imaging the sample consists of a narrow spectral band or several narrow spectral bands.

[0024] If the light imaging the sample is formed by one or more narrow spectral bands, the characteristic wavelength preferably lies in the middle of the respective narrow spectral band. Alternatively, preferably, a lower or upper end of the respective spectral band can also be considered the characteristic wavelength.

[0025] In preferred embodiments, a reference wavelength of the microscope is assumed. This reference wavelength is preferably the wavelength for which the microscope was optimized during its technical design, such that the spherical aberration is minimal for this wavelength even without correction. However, the invention also allows for minimizing the spherical aberration for other applications with different characteristic wavelengths. The data describing the spherical aberration as a function of wavelength are described in relation to this reference wavelength. Thus, the spherical aberration is described as a function of a deviation of the characteristic wavelength from the reference wavelength. This deviation is determined when determining the spectral range of the light imaging the sample through the microscope.The adjustment parameter of the correction tool is then specifically determined for the identified deviation.

[0026] In further preferred embodiments, the light imaging the sample through the microscope exhibits two or more spectral ranges, each preferably represented by a characteristic wavelength. These at least two spectral ranges occur simultaneously. When determining the control variable of the correction device based on the provided data describing the spherical aberration as a function of wavelength for the at least two identified spectral ranges, an optimum control variable suitable for the at least two spectral ranges is sought in order to minimize the spherical aberration simultaneously for the multiple spectral ranges.Since it is generally not possible to minimize the spherical aberration to zero for all spectral ranges when dealing with multiple spectral regions, a compromise must be found where the spherical aberration is as small as possible for all spectral regions. This search for an optimum is unnecessary if the light imaging the sample through the microscope does not exhibit the at least two spectral regions simultaneously, but rather sequentially. In this case, the spherical aberration can be minimized individually for each of the spectral regions.

[0027] In principle, the spectrum of the light imaging the sample through the microscope can also be a broadband or continuous spectrum. Accordingly, the adjustment parameter of the correction agent is determined based on the provided data describing the spherical aberration as a function of wavelength for at least one determined spectral range, such that the spherical aberration can be minimized across the determined spectral range.

[0028] In preferred embodiments, data describing the correction of the spherical aberration effected by the correction means as a function of the correction means's position are provided. This data is preferably stored in the microscope or the objective lens. This data is particularly necessary to determine the adjustment parameter of the correction means, based on the provided data describing the spherical aberration as a function of wavelength for the at least one determined spectral range, in order to minimize the spherical aberration for the at least one determined spectral range.Alternatively, and preferably, the spherical aberration to be corrected is used as an intermediate value, so that the step of determining the adjustment value of the correction device, based on the provided data describing the spherical aberration as a function of wavelength for the at least one determined spectral range, comprises two sub-steps. In one sub-step, the spherical aberration that would occur in the at least one determined spectral range if the correction device remained in its reference position is calculated. This is therefore the spherical aberration to be corrected. In another sub-step, the adjustment value of the correction device is calculated to minimize the calculated spherical aberration for the at least one determined spectral range.

[0029] In embodiments where the deviation of the characteristic wavelength from the reference wavelength is determined, the data used to describe the correction of the spherical aberration effected by the correction means as a function of the correction means's position are preferably used to determine the dependence of the correction means's position on this deviation. Thus, the control variable can be directly derived from the determined deviation. Alternatively, if the spherical aberration to be corrected is preferably used as an intermediate variable, data describing the dependence of the spherical aberration to be corrected on the deviation of the characteristic wavelength from the reference wavelength, as well as data describing the dependence of the correction means's position on the spherical aberration to be corrected, must be provided.

[0030] The position of the correction device is preferably described in the form of increments. These increments are preferably steps of the actuator of the correction device. The actuator is preferably a stepper motor.

[0031] The data described above, describing the various dependencies, is preferably stored in a data storage device located in the objective lens or another component of the microscope. Alternatively, the data can be stored in a cloud and / or on a control PC belonging to the microscope.

[0032] The aforementioned data describing the various dependencies are preferably derived from data provided by the microscope manufacturer and / or determined through machine learning based on the microscope settings configured by the operator. Artificial intelligence can be used for this purpose.

[0033] In preferred embodiments, the microscope comprises a coverslip and / or a sample holder. The sample is placed on the sample holder. The coverslip is positioned over the sample. Preferably, an immersion medium is present between the objective lens and the coverslip or sample holder. The sample, the coverslip or sample holder, and / or the immersion medium influence the spherical aberration when the sample is examined under the microscope. Therefore, in preferred embodiments, additional data are provided to describe the correction of the spherical aberration effected by the correction device as a function of the correction device's position and as a function of at least one sample space parameter.The at least one sample space parameter is formed by a thickness of the cover or support glass, a refractive index of the cover or support glass, a penetration depth of the light into the sample, a refractive index of the sample, a thickness of the immersion medium, a refractive index of the immersion medium and / or a temperature of a sample space surrounding the sample.The determination of the adjustment parameter of the correction agent is carried out based on the provided data for describing the spherical aberration as a function of the wavelength and based on the data for describing the correction of the spherical aberration caused by the correction agent as a function of the position of the correction agent and as a function of the at least one sample space parameter, in order to correct the spherochromatic aberration for the at least one determined spectral range and also an aberration caused by the at least one sample space parameter.

[0034] In preferred embodiments, the data describing the correction of the spherical aberration effected by the correction means as a function of the position of the correction means and as a function of the at least one sample space parameter also describe an interaction of a variation of the at least one sample space parameter with the correction of the spherochromatic aberration. This ensures that the interaction of the variation of the at least one sample space parameter with the chromatic correction is taken into account when calculating the position of the correction means.

[0035] In preferred embodiments, the method comprises a further step. The lens focus is adjusted to the optical image of the lens, which has changed due to the setting of the correction means to the control variable. Setting the correction means to the control variable can lead to a defocusing of the lens, which is corrected by the aforementioned step. This preferably occurs automatically and continuously.

[0036] The microscope according to the invention is used for microscopic examination of a sample. The microscope comprises an objective lens and a correction device arranged in a beam path of the microscope for correcting a spherical aberration. The microscope also includes a control unit configured to carry out the method according to the invention. The control unit is preferably configured to carry out one of the preferred embodiments of the method according to the invention described above. Furthermore, the microscope preferably also has features described in connection with the method according to the invention.

[0037] Further details and developments of the invention will become apparent from the following description of preferred embodiments of the invention in comparison to the prior art, with reference to the drawing. The drawing shows: Fig. 1: a diagram illustrating the optical quality of a first exemplary lens optimized according to the state of the art 1; Fig. 2: a diagram illustrating the optical quality of the first exemplary lens 1 optimized according to the invention; Fig. 3: a diagram illustrating the optical quality of a second exemplary lens 2 optimized according to the state of the art; and Fig. 4: a diagram illustrating the optical quality of the second exemplary lens optimized according to the invention.

[0038] Fig. Figure 1 shows a diagram illustrating the optical quality of a first exemplary objective 1 optimized according to the prior art. The objective 1 (not shown) is used in a microscope (not shown). The objective 1 includes a correction ring (not shown) for correcting a spherical aberration. According to the prior art, the objective 1 is typically optimized for the green region of the spectrum, for example, at a wavelength of 546 nm, which can also be considered a reference wavelength. The correction ring (not shown) is adjusted so that the spherical aberration at this reference wavelength is almost negligible. In the exemplary prior art optimization for 546 nm, the residual spherical aberration at a wavelength of 546 nm is only -0.02, whereas at a wavelength of 850 nm it has a value of +0.55.

[0039] The optical quality of the optimized first lens 1 is represented in the diagram by the Strehl ratio, which is plotted dimensionlessly on the y-axis. The x-axis of the diagram represents the distance from the optical axis in the image, in millimeters. The first graph, 01, shows the Strehl ratio at a wavelength of 405 nm. The second graph, 02, shows the Strehl ratio at a wavelength of 435 nm. The third graph, 03, shows the Strehl ratio at a wavelength of 480 nm. The fourth graph, 04, shows the Strehl ratio at a wavelength of 546 nm. The fifth graph, 05, shows the Strehl ratio at a wavelength of 643 nm. The sixth graph, 06, shows the Strehl ratio at a wavelength of 850 nm. At a wavelength of 850 nm, the Strehl ratio is approximately 87% on the optical axis and approximately 55% in the field at 12 mm.

[0040] Fig. Figure 2 shows a diagram illustrating the optical quality of the first exemplary lens 1 optimized according to the invention. According to the method of the invention, the spherical aberration is minimized for a characteristic wavelength of, for example, 850 nm. In the optimization carried out according to the invention for, for example, 850 nm, the residual spherical aberration is minimized to 0 at a wavelength of 850 nm, whereas it has a value of -0.35 at a wavelength of 546 nm.

[0041] The diagram shows how in Fig. Figure 1 shows the Strehl ratio of the optimized first lens 1. A seventh graph 11 shows the Strehl ratio at a wavelength of 405 nm. An eighth graph 12 shows the Strehl ratio at a wavelength of 435 nm. A ninth graph 13 shows the Strehl ratio at a wavelength of 480 nm. A tenth graph 14 shows the Strehl ratio at a wavelength of 546 nm. An eleventh graph 15 shows the Strehl ratio at a wavelength of 643 nm. A twelfth graph 16 shows the Strehl ratio at a wavelength of 850 nm. The Strehl ratio at a wavelength of 850 nm is approximately 98% on the optical axis and approximately 70% in the field at 12 mm, and is therefore significantly better than that of the lens shown in Figure 1. Fig. 1. Optimization shown according to the state of the art.

[0042] Fig. Figure 3 shows a diagram illustrating the optical quality of a second exemplary objective 2 optimized according to the prior art. The objective 2 (not shown) is used in a microscope (not shown). The objective 2 also includes a correction ring (not shown) for correcting a spherical aberration. According to the prior art, the objective 2 is typically optimized in the green region of the spectrum, for example, at a wavelength of 546 nm, which can also be considered a reference wavelength. The correction ring (not shown) is adjusted so that the spherical aberration is largely eliminated at this reference wavelength. In the exemplary prior art optimization for 546 nm, the residual spherical aberration at a wavelength of 546 nm was minimized to 0, whereas at a wavelength of 850 nm it has a value of +1.73.

[0043] The optical quality of the second object 2, optimized according to the state of the art, is shown in the diagram as in Fig. 1. The Strehl ratio is represented by the Strehl ratio. A thirteenth graph, 21, shows the Strehl ratio at a wavelength of 405 nm. A fourteenth graph, 22, shows the Strehl ratio at a wavelength of 435 nm. A fifteenth graph, 23, shows the Strehl ratio at a wavelength of 480 nm. A sixteenth graph, 24, shows the Strehl ratio at a wavelength of 546 nm. A seventeenth graph, 25, shows the Strehl ratio at a wavelength of 643 nm. An eighteenth graph, 26, shows the Strehl ratio at a wavelength of 850 nm. The Strehl ratio at a wavelength of 850 nm is approximately 30% on the optical axis.

[0044] Fig. Figure 4 shows a diagram illustrating the optical quality of the second exemplary lens 2 optimized according to the invention. According to the method of the invention, the spherical aberration is minimized for a characteristic wavelength of, for example, 850 nm. In the optimization carried out according to the invention for, for example, 850 nm, the residual spherical aberration is minimized to 0 at a wavelength of 850 nm, whereas it has a value of -0.91 at a wavelength of 546 nm.

[0045] The diagram shows how in Fig. Figure 3 shows the Strehl ratio of the optimized second lens 2. A nineteenth graph 31 shows the Strehl ratio at a wavelength of 405 nm. A twentieth graph 32 shows the Strehl ratio at a wavelength of 435 nm. A twenty-first graph 33 shows the Strehl ratio at a wavelength of 480 nm. A twenty-second graph 34 shows the Strehl ratio at a wavelength of 546 nm. A twenty-third graph 35 shows the Strehl ratio at a wavelength of 643 nm. A twenty-fourth graph 36 shows the Strehl ratio at a wavelength of 850 nm. The Strehl ratio at a wavelength of 850 nm on the optical axis is approximately 97% and is therefore significantly better than that of the lens shown in Figure 2. Fig. 3 optimizations shown according to the state of the art. Reference symbol list 01 first graph (405 nm) 02 second graph (435 nm) 03 third graph (480 nm) 04 fourth graph (546 nm) 05 fifth graph (643 nm) 06 sixth graph (850 nm) 07 - 08 - 09 - 10 - 11 seventh graph (405 nm) 12 eighth graph (435 nm) 13 ninth graph (480 nm) 14 tenth graph (546 nm) 15 eleventh graph (643 nm) 16 twelfth graph (850 nm) 17 - 18 - 19 - 20 - 21 Thirteenth graph (405 nm) 22 Fourteenth graph (435 nm) 23 Fifteenth graph (480 nm) 24 Sixteenth graph (546 nm) 25 Seventeenth graph (643 nm) 26 Eighteenth graph (850 nm) 27 - 28 - 29 - 30 - 31 nineteenth graph (405 nm) 32 twentieth graph (435 nm) 33 twenty-first graph (480 nm) 34 twenty-second graph (546 nm) 35 twenty-third graph (643 nm) 36 Twenty-fourth graph (850 nm) QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] JP 4554174 B2

[0003] US 9,741,122 B2

[0004] US 9,411,142 B2

[0005] EP 3 118 665 B1

[0006] WO 2009 / 096522 A1

[0007] US 6,563,634 B2

[0008] EP 3 112 919 A1

[0009] DE 10 2018 126 000 A1

[0010] DE 10 2018 126 021 B3

[0011] DE 10 2018 126 007 A1

[0012]

Claims

[1] Method for correcting a spherochromatic aberration that occurs when examining a sample with a microscope, wherein the microscope comprises an objective and a corrective means arranged in a beam path of the microscope for correcting a spherical aberration; and wherein the method comprises the following steps: - Providing data to describe the spherical aberration as a function of a wavelength of light imaged by the microscope; - Microscopy of a sample, whereby at least one spectral range of a light imaging the sample through the microscope is determined; - Determining a control parameter of the correction agent based on the provided data to describe the spherical aberration as a function of wavelength for the at least one determined spectral range, in order to minimize the spherical aberration for the at least one determined spectral range; and - Setting the correction factor to the determined control variable. [2] Method according to claim 1, characterized by , that at least one spectral range is determined from: - a spectrum of excitation radiation, - a spectrum of emission radiation shifted by a Stokes shift, - a spectrum of a color filter used for microscopy, - a spectrum of a dye used for microscopy, and / or - a measured spectrum of the imaging light. [3] Method according to claim 1 or 2, characterized by, that at least one spectral range of the light imaging the sample through the microscope is determined by determining a wavelength characteristic for each spectral range. [4] Method according to claim 3, characterized by , that the data for describing the spherical imaging error are described as a function of the wavelength in relation to a reference wavelength of the microscope, wherein when determining the characteristic wavelength of the at least one spectral range of the light imaging the sample by the microscope, a deviation of the characteristic wavelength from the reference wavelength is determined, and wherein the control variable of the correction mean for the determined deviation is determined. [5] Method according to any one of claims 1 to 4, characterized by, that at least two of the spectral ranges of the light imaging the sample through the microscope are determined, wherein, when determining the control variable of the correction agent, starting from the data provided to describe the spherical imaging error as a function of the wavelength, an optimum of the control variable applicable to the at least two determined spectral ranges is sought in order to minimize the spherical imaging error for the at least two determined spectral ranges. [6] Method according to any one of claims 1 to 5, characterized by , that data are provided to describe a correction of the spherical aberration caused by the correction agent as a function of a position of the correction agent. [7] Method according to any one of claims 1 to 6, characterized by, that the step of determining the control variable of the correction agent based on the provided data for describing the spherical imaging error as a function of wavelength for the at least one determined spectral range comprises the following sub-steps: - Calculating the spherical aberration that would occur in at least one determined spectral range; and - Calculating the adjustment value of the correction medium to minimize the calculated spherical imaging error for at least one determined spectral range. [8] Method according to any one of claims 1 to 7, characterized by, that data are provided to describe a correction of the spherical imaging error caused by the correction agent as a function of a position of the correction agent and as a function of at least one sample space parameter; wherein the at least one sample space parameter is formed by: - the thickness of a coverslip or support glass used for microscopic examination of the sample, - a refractive index of a coverslip or support glass used for microscopy of the sample, - the penetration depth of the light into the sample, - a refractive index of the sample, - the thickness of an immersion medium located above a cover or support glass, - a refractive index of an immersion medium located above a cover or support glass, and / or - a temperature of a sample chamber surrounding the sample; wherein the determination of the control variable of the correction agent is carried out based on the provided data for describing the spherical aberration as a function of the wavelength and based on the data for describing the correction of the spherical aberration caused by the correction agent as a function of the position of the correction agent and as a function of the at least one sample chamber parameter, in order to correct the spherochromatic aberration for the at least one determined spectral range and an aberration caused by the at least one sample chamber parameter. [9] Method according to claim 8, characterized by, that the data describing the correction of the spherical aberration caused by the correction agent as a function of the position of the correction agent and as a function of the at least one sample space parameter also describe an interaction of a variation of the at least one sample space parameter with the correction of the spherochromatic aberration. [10] Method according to any one of claims 1 to 9, characterized by that it includes the following further step: - Adjusting the focus of the lens to a change in the optical image of the lens caused by setting the correction medium to the control variable. [11] Method according to claim 10, characterized by , that the adjustment of the lens focus is automatic and continuous. [12] Microscope for microscopy of a sample, comprising an objective and a correction means arranged in a beam path of the microscope for correcting a spherical imaging error, wherein the microscope further comprises a control unit configured to carry out a method according to any one of claims 1 to 11.

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