Method for correcting a spherical aberration of a microscope and microscope

Description

[0001] The invention relates to a method for correcting a spherical aberration of a microscope having an objective lens and a cover glass or support glass, in which a correction means arranged in the objective lens is provided for correcting the spherical aberration. The invention further relates to a microscope having an adjustment device for correcting a spherical aberration.

[0002] Sample-related aberrations, especially refractive index mismatch, are the primary influence on the quality of a light microscopic image of a sample. The main causes of refractive index mismatch in biological microscopy are an inappropriate optical thickness of the coverslip and an inappropriate refractive index of the optical medium in which the sample is embedded.

[0003] A variety of lenses with correction means are known from the state of the art, which can be used to compensate for sample-induced spherical aberrations.

[0004] US 6,563,634 B2 discloses a microscope with a corrective lens for correcting imaging errors, in particular the thickness of a cover glass, a Petri dish, or a slide. US 6,563,634 B2 also discloses a method for correcting the position of a focal plane of the objective using the corrective lens.

[0005] US Pat. No. 9,411,142 B2 discloses a method for adjusting a correction ring when focusing a microscope. The method is based on the interpolation of support points determined by correction ring settings that must be defined by an operator. Using this method for samples with a large refractive index variation, the course of the correction ring adjustment can be adapted to the sample. However, precise evaluation and thus the determination of the support points is complex and requires an experienced operator. Furthermore, the aforementioned adjustment requires the generation of a large number of images, which is particularly disadvantageous for light-sensitive samples, e.g., due to fluorescence bleaching or phototoxicity.

[0006] US 2005 / 0024718 A1 discloses a microscope with an objective lens having a correction lens and an adjustment device for adjusting the correction lens. The adjustment device adjusts the correction lens based on a refractive index of a sample entered by the operator and a temperature measured in a sample chamber.

[0007] Document EP 1 988 417 A1 discloses a microscope with an objective lens having a correction ring. The document further discloses a method for correcting an aberration caused by variations in the optical thickness of a cover glass.

[0008] Regarding the state of the art, reference is also made to DE 10 2017 105 928 A1, which discloses a microscope and method for imaging an object.

[0009] It is an object of the invention to provide a method for correcting a spherical aberration of a microscope and a microscope with an adjustment device for correcting a spherical aberration, which enable a correction taking into account measurable properties of the sample and the sample space.

[0010] This object is achieved by a method having the features of claim 1 and by a microscope having the features of claim 9. Advantageous further developments can be found in the dependent claims.

[0011] The method according to the invention serves to correct a spherical aberration of a microscope with an objective and a cover or support glass, in which a correction means arranged in the objective is provided for correcting the spherical aberration. The microscope determines the refractive index of an optical medium adjacent to the cover or support glass and the thickness of the cover or support glass along the optical axis of the objective. The spherical aberration is determined based on the refractive index of the optical medium and the thickness of the cover or support glass. Based on the spherical aberration, a control variable is determined, based on which the correction means is adjusted such that the spherical aberration is corrected.

[0012] In this application, cover glass or support glass is understood to mean in particular a cover glass, a microscope slide, a bottom of a Petri dish or a bottom of a well of a microtiter plate.

[0013] According to the invention, the microscope determines properties of the sample to be imaged and the sample chamber, in particular the refractive index of the optical medium and the thickness of the cover glass or support glass. The spherical aberration is then determined from the properties determined by the microscope. The determined spherical aberration is corrected or compensated by adjusting the correction means.

[0014] The determination of the properties of the sample to be imaged and the sample space by the microscope itself, i.e. in-situ, is advantageous compared to known methods in which the imaging error is determined ex-situ, for example using a reference preparation, since no correlation can be ensured between the imaging error determined using the reference preparation and the imaging error when imaging the sample.

[0015] The aberration is determined, for example, by modeling a wavefront error from the properties of the sample and the sample space determined by the microscope. Models for wavefront errors based on properties of the sample and the sample space are known, for example, from P. Török et al., "Electromagnetic diffraction of light focused through a planar interface between materials of mismatched refractive indices: an integral representation," J. Opt. Soc. Am. A 12 (1995), pp. 325-332. Based on the determined spherical aberration, the correction means can be adjusted such that a further spherical aberration induced by the correction means compensates for the determined spherical aberration, thereby correcting the determined spherical aberration.

[0016] In a preferred embodiment, the control variable on the basis of which the correction means is adjusted is determined by minimizing a total image error formed by the spherical image error and a known further spherical image error induced by the correction means.

[0017] In a preferred embodiment, the microscope determines a mechanical distance between the objective lens and an object plane, and the spherical aberration is determined based on this mechanical distance. The mechanical distance can be determined, for example, using a coded or automated focus drive of the objective lens as soon as the position of the sample-facing surface of the cover glass or support glass is known. This position can be determined, for example, using a suitable autofocus system. Alternatively, the mechanical distance between the objective lens and the object plane can be entered by an operator.

[0018] In a particularly preferred embodiment, the determination of the mechanical distance between the lens and the object plane, the determination of the spherical aberration, and the determination of the manipulated variable on the basis of which the correction means is adjusted are performed repeatedly for the object plane and / or other object planes. In particular, the determination of the mechanical distance between the lens and the object plane, the determination of the spherical aberration, and the determination of the manipulated variable are repeated in a continuous loop while the lens is being adjusted to the object plane. This makes it possible to correct the spherical aberration even during a focusing process of the lens.

[0019] Preferably, the spherical aberration is determined based on the refractive index of the cover or support glass. The refractive index of the cover or support glass is either known in advance—the refractive indices of various cover glasses are standardized, for example, in ISO 8255-1—or is determined ex situ and then input into the microscope by an operator. Particularly preferably, the thickness of the cover or support glass for determining the spherical aberration is determined by the microscope itself.

[0020] Preferably, an immersion medium is placed between the objective lens and the cover glass or support glass. The spherical aberration is determined based on the refractive index of the immersion medium. The refractive index of the immersion medium can be determined, in particular, by the microscope itself.

[0021] In a preferred embodiment, to determine the manipulated variable, based on which the correction means is adjusted, at least one value of the manipulated variable and a value of the spherical aberration associated with the manipulated variable are provided by the lens. The value of the manipulated variable can, for example, be calculated from optical data of the lens or determined interferometrically using an individual lens.

[0022] In a particularly preferred embodiment, the value of the spherical aberration associated with the value of the manipulated variable is provided as expansion coefficients of orthogonal polynomials, in particular Zernike polynomials. This is a particularly simple way to quantify the spherical aberration.

[0023] The invention further relates to a microscope comprising an objective lens with a correction means that can be adjusted to correct a spherical aberration, and a cover or support glass. The microscope has an adjustment device for adjusting the correction means. The adjustment device is designed to determine the refractive index of an optical medium adjacent to the cover or support glass and / or the thickness of the cover or support glass along the optical axis of the objective lens, to determine a spherical aberration based on the refractive index of the optical medium and / or the thickness of the cover or support glass, and to determine a manipulated variable based on the spherical aberration, by means of which the correction means can be adjusted such that the spherical aberration is corrected.

[0024] Preferably, the correction means comprises at least one lens which is movable along the optical axis of the objective to correct imaging errors.

[0025] They show: Figure 1 shows a schematic representation of a microscope as an embodiment; Figure 2 shows a flow chart showing an embodiment of the method according to the invention for correcting a spherical aberration using the microscope according to Figure 1 shows.

[0026] Figure 1shows a microscope 10 as an exemplary embodiment. The microscope 10 has an objective 12 directed onto a cover glass 16. The objective 12 includes a correction means 14, which is adjustable for correcting an imaging error and is embodied, for example, as a lens that is displaceable along the optical axis O of the objective 12. The microscope 10 further includes a control unit 18, which is connected to the objective 12 via an external cable 20 and which controls the various objective components.

[0027] On the cover glass 16 is an optical medium 22 in which a sample is embedded, which is referred to below as the embedding medium 22. An immersion medium 24 is also inserted between the objective lens 12 and the cover glass 16.

[0028] Figure 2shows a flow chart illustrating an embodiment of the method according to the invention for correcting a spherical aberration using the microscope 10 according to Figure 1 shows.

[0029] In a first step S1, the thickness of the cover glass 16 is determined along the optical axis O of the objective lens 12. For this purpose, a measuring light beam is directed, for example, at oblique incidence through the objective lens 12 onto the cover glass 16. Partial reflections of the measuring light beam at a first optical interface formed by the cover glass 16 and the immersion medium 24 and at a second optical interface formed by the cover glass 16 and the embedding medium 22 generate two spatially separated reflected light beams. The two reflected light beams are received by the objective lens 12 and directed onto a position-sensitive detector. The thickness of the cover glass can be determined based on the detected locations of incidence of the two reflected light beams on the position-sensitive detector.

[0030] In a second step S2, the refractive index of the embedding medium 22 is determined. This can be done, for example, by detecting the intensities of the two reflected light beams generated in the first step S1 using the position-sensitive detector. Based on the detected intensities of the two reflected light beams, the refractive index of the embedding medium 22 can be determined, since the intensities of the two reflected light beams depend on the reflection and transmission of the measuring light beam at the two interfaces defined by the cover glass 16 and the embedding medium 22 or the immersion medium 24. The reflection and transmission processes, on which the intensities of the two spatially separated reflected light beams are ultimately based, are thus essentially determined by the refractive indices of the cover glass 16 as well as the embedding medium 22 and the immersion medium 24.

[0031] In a third step S3, a spherical aberration is determined from the thickness of the cover glass 16 along the optical axis O of the objective and the refractive index of the embedding medium 22.

[0032] From the refractive index of the embedding medium 22 bordering the cover glass 16 and the thickness of the cover glass 16 along the optical axis O of the objective 12, the imaging error in the form of a wavefront error can be determined as ψ r = z n Spc 2 − r 2 NA 2 + Δ d n Dgl 2 − r 2 NA 2 − z + Δ d n Imm 2 − r 2 NA 2

[0033] Where r is a radial coordinate of the exit pupil of the lens 12 normalized to the unit circle, z is a mechanical focal length, n spc the refractive index of the embedding medium 22 adjacent to the cover glass 16, n Imm the known refractive index of the immersion medium 24 introduced between the objective and the cover glass, Δd the deviation of the cover glass thickness along the optical axis O of the objective 12 from the nominal thickness of the cover glass 16 and N / Athe numerical aperture of the lens 12.

[0034] In a fourth step S4, a mechanical distance between the objective 12 and the coverslip 16 is determined using a coded focus drive of the objective 12 based on the position of a sample-facing surface of the coverslip 16. This position can be determined, for example, using a suitable autofocus system. Alternatively, the mechanical distance can be entered by an operator.

[0035] In a fifth step S5, a manipulated variable is determined based on the spherical aberration determined in the third step S3, which is used to adjust the correction means 14 such that the spherical aberration is corrected. The manipulated variable is determined by calculating a sum—hereinafter also referred to as the total aberration—of the spherical aberration determined in the third step S3 and a further spherical aberration induced by the correction means 14, which is a function solely of the manipulated variable. The sum is then minimized by minimizing the root mean square of this sum over the entire pupil. The total aberration is thus also a function solely of the manipulated variable.If both the determined spherical aberration and the spherical aberration induced by the correction agent are expanded up to the Nth order according to Zernike polynomials, the root mean square of the total aberration can be written as . ∑ k = 1 N A k ϕ k c + ψ k z , n Spc , Δ d 2

[0036] These include Ak Normalization constants, ψ k the coefficients of the Zernike polynomials of the determined spherical aberration, where k = 0 corresponds to the coefficient of the Zernike polynomial Z 0 0 corresponds, k = 1 the Zernike polynomial Z 2 0 etc. and ϕ k the coefficients of the Zernike polynomials of the spherical aberration induced by the correction means 14, which depend on the control variable denoted by c. Preferably, the component of the pure defocus ψ d r = 1 n Imm 2 n Imm 2 − NA 2 removed from the expansion coefficients. By differentiating equation (2) we obtain the following condition for an extremum 0 = ∑ k = 1 N A k dϕ k c dc ϕ k c + ψ k z , n Spc , Δ d

[0037] Equation (3) can now be calculated according to the following c opt The optimal value of the manipulated variable, referred to as [ ], can be resolved, based on which the correction means 14 can be adjusted so that the spherical aberration is corrected or compensated. This can be done, for example, using iterative numerical methods, such as those known from "Numerical Recipes, The Art of Scientific Computing", Cambridge University Press, 3rd edition, 2007, pages 487 to 502, ISBN 0-521-88068-8. For the case of a series expansion up to the fourth order (N = 4), c opt even analytically. Especially in the case that the coefficients ϕ k the Zernike polynomials of the spherical aberration induced by the correction means 14 depend only linearly on the manipulated variable or can be linearized in a suitable manner, the following solution is obtained c opt = − ∑ k = 1 N A k ψ k z , n Spc , Δ d ∑ k = 1 N A k ϕ k

[0038] Preferably, a series expansion of the determined spherical aberration and the spherical aberration induced by the correction means 14 is carried out up to the tenth order (N = 10). For this order, the necessary calculation operations are carried out both for a linear dependence of ϕ k ( c ) as well as for a non-linear dependency is numerically easy to handle and can also be implemented on simple components such as a PIC (programmable interrupt controller).

[0039] In a sixth step S6, the correction means 14 is adjusted to the manipulated variable determined in the fifth step S5 c opt This corrects the spherical aberration determined in the third step S3.

[0040] The fourth to sixth steps S4, S5, S6 are repeated for further object levels 12, as in Figure 2 is indicated by an arrow P1. List of reference symbols

[0041] 10Microscope 12Objective 14Correction medium 16Cover glass 18Control unit 20Cable 22Embedding medium 24Immersion medium P1Arrow

Claims

1. Method for correcting a spherical aberration of a microscope (10) with an objective (12) and a cover or support glass (16), in which a correction means, disposed in the objective (12), is provided for correcting the spherical aberration, characterized in that the refractive index of an optical medium (22, 24) bordering the cover or support glass (16) and / or the thickness of the cover or support glass (16) along the optical axis of the objective (12) is determined by the microscope (10), based on the refractive index of the optical medium (22, 24) and / or the thickness of the cover or support glass (16), the spherical aberration is determined, and based on the spherical aberration, a manipulated variable is determined, by means of which the correction means (14) is adjusted such that the spherical aberration is corrected, wherein the manipulated variable is determined mathematically, in which the root mean square of the sum of the spherical aberration and a further spherical aberration, induced by the correction means (14), is minimized over the entire pupil.

2. The method according to claim 1, characterized in that the determination of the manipulated variable on the basis of which the correction means (14) is adjusted is carried out by minimizing a total imaging error formed by the spherical imaging error and a known further spherical imaging error induced by the correction means (14).

3. The method according to claim 1 or 2, characterized in that a mechanical distance between the objective (12) and an object plane (12) is determined by the microscope (10) and the spherical aberration is determined based on this mechanical distance.

4. The method according to claim 3, characterized in that the determination of the mechanical distance between the objective (12) and the object plane (12), the determination of the spherical aberration and the determination of the manipulated variable on the basis of which the correction means (14) is adjusted, are carried out repeatedly for the object plane and / or further object planes (12).

5. The method according to any one of claims 1 to 4, characterized in that the determination of the spherical aberration is carried out based on the refractive index of the cover or support glass (16).

6. The method according to any one of claims 1 to 5, characterized in that an immersion medium is introduced between the objective (12) and the cover or support glass (16) and the determination of the spherical aberration is carried out based on the refractive index of the immersion medium.

7. The method according to any one of claims 1 to 6, characterized in that, in order to determine the manipulated variable on the basis of which the correction means (14) is adjusted, at least one value of the manipulated variable and a value of the spherical aberration associated with the value of the manipulated variable are provided by the objective (12).

8. The method according to claim 7, characterized in that the value of the spherical aberration, associated with the value of the manipulated variable, is provided as expansion coefficients of orthogonal polynomials, in particular Zernike polynomials.

9. Microscope, comprising: an objective (12) with a correction means (14) which is adjustable to correct a spherical aberration, and a cover or support glass (16), characterized in that the microscope (10) has an adjusting apparatus for adjusting the correction means (14), the adjusting apparatus is configured to determine the refractive index of an optical medium (22, 24) bordering the cover or support glass (16) and the thickness of the cover or support glass (16) along the optical axis of the objective (12), the adjusting apparatus is configured to determine a spherical aberration, based on the refractive index of the optical medium (22, 24) and the thickness of the cover or support glass (16), and the adjusting apparatus is configured to determine, based on the spherical aberration, a manipulated variable, by means of which the correction means (14) can be adjusted such that the spherical aberration is corrected, wherein the adjusting apparatus is further configured to determine the manipulated variable mathematically, in which the root mean square of the sum of the spherical aberration and a further spherical aberration, induced by the correction means (14), is minimized over the entire pupil.

10. The microscope according to claim 9, characterized in that the correction means (14) comprises at least one lens which is movable along the optical axis (O) of the objective (12) to correct imaging errors.