Objective, optical arrangement and microscope, in particular for use in the imaging of organoids

Abstract

The invention relates to an objective (3), in particular a detection objective for a microscope (1). The objective (3) has a plurality of optical elements arranged along an optical axis (AI) in the beam path of the objective (3). The invention is characterised in that a pupil (AP) is formed in the beam path outside a sequence of light passage surfaces of the optical elements along the optical axis (AI) and downstream thereof in the light direction. The invention further relates to an optical arrangement (21) having the objective (3) and to a microscope (1).

Description

[0001] Objective lens, optical arrangement and microscope, especially for use in imaging organoids

[0002] The invention relates to an objective lens, in particular a detection lens for a microscope, according to the preamble of the independent claim. The invention further relates to an optical arrangement comprising an objective lens, a microscope, and the use of the invention.

[0003] In the field of microscopy, in particular so-called light-sheet microscopy (selective-plane illumination microscopy, SPIM), objects to be observed and imaged (samples) are often illuminated or imaged using illumination beam paths and / or detection beam paths that are inclined relative to a sample plane in which the sample is arranged (see for example DE 10 2013 107 297 Al).

[0004] With regard to the available installation space and freedom from manipulation, inverse arrangements of, for example, detection optics are often used, in which an image is formed obliquely through a transparent component, for example a sample carrier such as a glass plate, a Petri dish, a multiwell plate or the like.

[0005] Due to the oblique passage of light rays through the sample holder, significant aberrations can occur. Several solutions to correct these are known from the prior art.

[0006] For example, DE 10 2013 112 595 Al proposes a detection correction lens in the form of a freeform lens as a component of a detection objective. Dynamic adjustments can be made, for example, by means of individual (DE 102020211 148 Al) or mutually movable correction elements with freeform surfaces (Alvarez plates) (for example, DE 10 2014 104977 Al, WO 2015 / 155027 Al, DE 102020211 148 Al).

[0007] From EP 2 149064 Bl, a revolver with a plurality of different optical elements is known, which can be selectively swung into the beam path of a detection objective in order to reduce aberrations that occur.

[0008] The possibilities proposed in the prior art involve high-resolution imaging systems with large numerical apertures and comparatively small image fields. For observing and imaging larger samples, such as organoids, the known technical solutions are less suitable, as they are, for example, expensive and time-consuming. Their use for mass screening is not efficiently possible.

[0009] Organoids are defined here as organ-like structures generated from pluripotent stem cells using cell culture methods. Although organoids lack blood vessels and stroma, they behave similarly to organs in many respects.

[0010] The invention aims to propose a method for observing and imaging samples that is more efficient and cost-effective than the prior art. At the same time, it should also provide a method for reducing aberrations.

[0011] The problem is solved by an objective lens according to the subject matter of the main claim. An optical arrangement, a microscope, and a use thereof are the subject matter of the respective dependent claims. The problem is solved by an objective lens, in particular a detection objective lens for a microscope, which has a plurality of optical elements arranged along an optical axis in the beam path of the objective lens.

[0012] A characteristic feature of a lens according to the invention is that a pupil is formed outside a sequence of light-transmitting surfaces of the optical elements along the optical axis and following them in the direction of light in the beam path. A lens according to the invention thus advantageously provides a pupil that offers sufficient space for optional manipulation of the light rays guided along the beam path.

[0013] To enable such manipulation, one embodiment of the invention provides a replaceable first correction element, which can be positioned manually or mechanically in or near the pupil, to reduce existing aberrations. Such aberrations occur particularly as a result of the oblique passage of radiation, which is to be captured by the objective lens, through a transparent sample carrier. The objective lens housing can have a corresponding opening to move the first correction element into or out of the beam path. The opening can advantageously be sealed light-tight when the first correction element is positioned in the beam path or when the objective lens is to be used without a correction element.

[0014] Advantageously, at least one light transmission surface of the first correction element is designed as a freeform surface. The advantage of such an embodiment of the invention lies in the ability to select the use of the first correction element as needed. The design with at least one freeform surface allows for a correction adapted to specific combinations of the optical properties of different samples, any markers used, sample carriers, detection angles, and lens properties.

[0015] According to the invention, the reduction of aberrations is achieved in particular by the action of the first correction element. The wavefronts of the detected radiation are modified in such a way that the desired improvement in image quality is achieved.

[0016] In an advantageous embodiment of the lens according to the invention, the freeform surface can be designed symmetrically to a plane of symmetry spanned by the optical axis and a normal to the optical axis.

[0017] The freeform surface, or rather the freeform surfaces, can be defined by an equation of the form The freeform surface can be described as follows: x, y, and z denote the three Cartesian coordinates of a point lying on the freeform surface. Only even powers with non-zero coefficients are used to describe the freeform surface in a direction orthogonal to the plane of symmetry.

[0018] In contrast, parallel to the plane of symmetry, both even and odd powers with non-zero coefficients are used.

[0019] For the purposes of this description, a coefficient is non-zero if it produces an optical effect above a predetermined tolerance threshold. This effect can be measured by the phase change caused by rays passing through a freeform surface. The thickness change of the freeform element caused by the corresponding coefficient can be multiplied by the refractive index (phase change) for all rays at all points within the optically used area. If the phase change is below a predefined tolerance threshold, the corresponding coefficient is considered to have essentially the same effect as zero. If the resulting phase change is greater than the tolerance threshold, the coefficient is considered non-zero. The tolerance threshold could be, for example, one-tenth, one-twentieth, or one-fortieth of the mean wavelength used.

[0020] In a simple embodiment of the lens according to the invention, the first correction element has one flat light transmission surface and one freeform light transmission surface. This simplifies the manufacture, testing, and adjustment of the first correction element.

[0021] In further embodiments of the invention, both light transmission surfaces of the first correction element can be designed as freeform surfaces. For example, a symmetrical division of the surface profile according to the formula above onto the light transmission surfaces, i.e., onto the front and back surfaces, of a correction element can ensure that the profile depths remain small, thus enabling or supporting photolithographic manufacturing. In this way, the profile depth can be kept, for example, less than 10 to 30 pm.

[0022] To reduce the occurrence of unwanted reflections and / or to simplify manufacturing, profile terms can be superimposed on both light-transmitting surfaces, largely compensating each other with respect to the beams transmitted by the first correction element. Nevertheless, reflections are reduced and / or directed in directions where they will not be detected further down the beam path.

[0023] The objective lens according to the invention can be used to image comparatively large samples such as organoids. In contrast, conventional light-sheet microscopy focuses on molecules (fluorescence markers or cell structures labeled with them) to be imaged. When imaging areas of existing organoids and similar samples, numerical apertures of less than or equal to 0.6 can be used due to their size. It has been shown that at numerical apertures < 0.6, even without further correction, such as adaptive correction, image aberrations occur at a nearly diffraction-limited level and with Strehl ratios greater than 90%. Accordingly, the numerical aperture of the objective lens according to the invention is less than or equal to 0.6 in one possible embodiment of the invention.

[0024] To increase the depth of field and reduce residual aberrations caused by variations in the optical properties of, for example, the sample and / or the sample holder, it is advantageous to select smaller numerical apertures, for example NA = 0.5 or 0.4. In an advantageous embodiment of the lens according to the invention, a manually or mechanically interchangeable aperture, limiting its effect, can be provided in or near the lens pupil.

[0025] The lens according to the invention can have a suitably designed slot or a position in the beam path provided for and accessible by an aperture. To store a number of possible apertures and to be able to select and quickly bring them into the beam path as needed, a changer (aperture changer) with a number of different apertures can be provided. The changer can, for example, be designed as an aperture changer plate on which a number of different apertures are formed as openings (holes) and / or recesses along a circular path. A desired aperture size can be placed in the beam path manually or by means of a controlled movement. The changer can optionally have a recess or hole that is not aperture-limiting and that can be placed in the beam path when no aperture effect is desired via the changer.The changer can advantageously be designed in a disc shape and be rotatable around a pivot axis. In other possible designs, the changer can, for example, be an elongated or curved plate with corresponding recesses or holes, which can be adjusted along a controlled sliding path.

[0026] Alternatively or in addition to an aperture and the first correction element, further correction elements may be present in the lens's optical path. These may, but do not necessarily have to, be interchangeable. Other correction elements can include, for example, adaptive mirrors, deformable refractive components, or spatial light modulators (SLMs).

[0027] It is also possible, for example, that in one embodiment of the lens according to the invention, axial distances between at least two optical elements designed as rotationally symmetric lenses are adjustable. The optical elements can be designed to correct rotationally symmetric wavefront components.

[0028] In a further embodiment of a lens according to the invention, it is designed such that an image field of at least 0.8 mm, preferably at least 0.9 mm, and preferably at least 1 mm can be captured on the object side. A correspondingly large image field facilitates the use of the lens for imaging large samples, particularly organoids.

[0029] The lens according to the invention, in one of its embodiments, can be part of an optical arrangement. This arrangement comprises, in addition to the lens, a sample stage with a support surface for holding a sample, the support surface defining a sample plane. The optical axis of the lens is directed at an angle (detection angle) greater than 0° into the sample plane. The detection angle is the smaller of the two angles measured between the optical axis and a surface normal located on the sample plane. Detection radiation emanating from the sample thus passes obliquely through the sample support and is then collected by the lens according to the invention.

[0030] In another embodiment, the freeform surface can be symmetrical to a meridional plane spanned by the optical axis of the lens and a surface normal of the sample plane.

[0031] The objective lens and / or the optical arrangement according to the invention can be part of a microscope, in particular a light-sheet microscope. This can be advantageously used for area-by-area imaging of organoids.

[0032] The invention offers a number of technical advantages. For example, it provides a lens with an accessible pupil, thus enabling the use of an interchangeable correction element. Through the action of the correction element, aberrations that occur as a result of the oblique passage of the rays to be captured through a sample carrier can be reduced.

[0033] In contrast to the lens according to the invention, lenses known from the prior art are designed for a high numerical aperture in the range of approximately 0.95 and have a rather small field of view, typically 300 to 360 pm in diameter. A lens according to the invention has a numerical aperture of preferably no more than 0.6, but enables a field of view diameter of 1 mm and larger. This makes the lens advantageously suited for observing and imaging large samples, particularly organoids. Organoids can, for example, serve as laboratory models for human or animal organs such as the heart, stomach, intestines, kidneys, brain, retina, etc., in pharmaceutical research. They are also rapidly gaining importance in the study of diseases caused by viruses, parasites, or noxious agents, as well as in the study of the origin and treatment of idiopathic diseases such as cancer and Alzheimer's disease.They are already used today as a replacement for animal testing in efficacy tests for new drugs, as well as in determining limit values ​​for environmental toxins. Future applications include the production of artificial replacement organs (e.g., myocardial cells or artificial retinas) in the laboratory.

[0034] Since the typical refractive index of organoids is somewhat higher than that of individual cells (typically in the range of 1.34 to 1.38, in some rarer cases up to 1.4), an immersion medium to be used with the objective according to the invention can be adapted to a somewhat higher average refractive index of approximately 1.36. Ethanol, which is readily available and inexpensive, can advantageously be used as the immersion medium.

[0035] The dispensing with an adjustable wavefront manipulator, for example two controlled adjustable Alvarez plates (e.g. DE 102020 211 148 Al), allows for a more cost-effective construction of the lens according to the invention as well as lower requirements for adjustment.

[0036] The exemplary embodiments of the invention demonstrate that, despite significantly reduced complexity and manufacturing costs, a substantially diffraction-limited light-sheet microscope can be provided for organoid research. The invention is suitable, for example, for use in instruments employed for mass screenings where large numbers of samples need to be examined at low cost.

[0037] The invention is explained in more detail below with reference to illustrations and exemplary embodiments. These show:

[0038] Fig. 1 shows a schematic representation of an embodiment of a device according to the invention.

[0039] Lens;

[0040] Fig. 2 shows a schematic representation of an embodiment of a device according to the invention.

[0041] microscope;

[0042] Fig. 3 shows a table with results of a wavefront analysis of 13 selected

[0043] Field points of an example lens not shown;

[0044] Fig. 4 Representations of residual wavefront errors in units of the reference wavelength

[0045] 546.074 nm for field points Fl to F5;

[0046] Fig. 5 Representations of residual wavefront errors in units of the reference wavelength

[0047] 546.074 nm for field points F6 to F10; and

[0048] Fig. 6 Representations of residual wavefront errors in units of the reference wavelength

[0049] 546.074 nm for field points Fll to F13.

[0050] In the following schematic representations of exemplary embodiments of the invention, the same reference numerals denote the same elements. The lens group 9.1, or the optical lenses 9.11 and 9.12, are shown schematically with uniform biconvex lens shapes, regardless of their actual shape. An objective 3 according to the invention may have a different number of optical elements.

[0051] A simplified embodiment of a lens 3 according to the invention, particularly in the form of a detection lens 3 with a first optical axis Al, is shown schematically in Fig. 1 as a longitudinal section through the lens 3. Optical lenses 9.11 to 9.12 and a first correction element 12 are arranged along the first optical axis Al of the lens 3. In this embodiment, the correction element 12 has a planar surface and a freeform surface. In other possible embodiments, the first correction element 12 can be designed as a freeform surface on both light-transmitting surfaces facing the beam path. The first correction element 12 is arranged sufficiently close to the plane of a pupil P. A plane in which the beams overlap by at least 80%, preferably at least 90%, and preferably at least 95% is considered sufficiently close.This is achieved by means of a corresponding optical design (construction) of the lens 3 after the first lens group 9.1, thereby providing sufficient installation space for the first correction element 12 and any additional mechanical components (not shown).

[0052] By means of a drive 14, the first correction element 12 can be brought into the beam path of the objective 3 by positioning the first correction element 12 on the first optical axis Al. Similarly, the first correction element 12 can be removed from the beam path. A detection beam DS (see Fig. 2) captured by the objective 3 and guided along the beam path is imaged by the action of the optical lenses 9.11 and 9.12. The wavefronts of the detection beam DS are advantageously influenced by the action of the first correction element 12 in such a way that aberrations are reduced, which are caused in particular by an oblique passage of beams through a sample carrier 7 (see Fig. 2).

[0053] The ability to change the first correction element 12 allows for adaptation to different optical requirements when using the lens 3 according to the invention. Optionally, a magazine 19 can be provided for this purpose, in which differently designed first correction elements 12 are stored. Using suitable mechanics and control via a controller 13, a selected first correction element 12 can be removed from the magazine 19 and placed in the beam path of the lens 3. A first correction element 12 that is no longer needed can be (re)inserted into the magazine 19. The process of selecting the correction element 12, changing the correction element 12, and / or removing it from or inserting it into the magazine 19 can be performed manually or automatically.

[0054] Facing a sample 5 (see Fig. 2) and positioned upstream of the first correction element 12 is the first lens group 9.1, which in this exemplary embodiment is formed by the front lens 9.11 and an optical lens 9.12. An optional aperture diaphragm 10 is located downstream of the correction element 12 in the beam path. The first correction element 12 and the aperture diaphragm 10 are arranged near the pupil P of the objective 3.

[0055] The aperture diaphragm 10 itself is, for example, configured as an opening 22 or a recess 22 of an aperture changer 20, which is advantageously designed as an aperture changer plate. The aperture changer 20 has a number of openings 22 or recesses 22 of different sizes along a circular path and is rotatable about a rotational axis (indicated) by means of a drive 23. If required, a desired aperture diaphragm 10 can thus be selected and positioned in the beam path. For example, numerical apertures of the lens 3 of 0.6, 0.5, 0.4, or 0.3 can be set in this way.

[0056] The drives 14 and 23 can be controlled by control commands from the control unit 13. They can be, for example, electric drives or piezoelectric drives.

[0057] Figure 2 shows an embodiment of an optical arrangement 21 according to the invention, comprising the objective 3 and a sample stage 11 with a support surface for holding a sample 5, wherein a sample plane 4 is defined by the support surface. The optical arrangement 21 is part of an inverted microscope 1. The microscope 1 has an illumination objective 2 and a detection objective 3. By means of the illumination objective 2 and an illumination beam BS, a light sheet 6 is generated or can be generated along a second optical axis A2, which can be used to examine a sample 5 arranged in a sample plane 4. The detection objective 3 has an optical axis Al, along which detection light DS coming from the sample plane 4 is directed onto a detector 18 and can be detected by it.The first optical axis Al and the second optical axis A2 are orthogonally aligned to each other and each encloses an angle of 45° (detection angle and illumination angle, respectively) with a surface normal N of the sample plane 4, which serves as the reference plane. In a further embodiment described below, the detection angle is 25°, while the illumination angle is 65°.

[0058] Sample 5 is arranged in a Petri dish, serving as a sample holder 7, located on a sample stage 11. The Petri dish is filled with a liquid, forming a medium 8 surrounding the sample 5. The sample holder 7 is optionally held on the sample stage 11. The sample stage 11 itself is adjustable by means of drives (not shown).

[0059] In the illustrated embodiment, the detection objective 3 is provided with the first correction element 12. The first correction element 12 serves to correct aberrations that can occur due to the oblique passage of the illumination radiation BS through the base of the sample carrier 7.

[0060] The correction element 12 is connected to an adjustment device in the form of a motor drive 14, which can be controlled by means of a control unit 13 (only indicated).

[0061] The illumination radiation BS is provided by a laser module 15 and shaped by a beam shaping unit 16. The beam shaping unit 16 is, for example, an optic by which the provided illumination radiation BS is collimated. In further embodiments, the beam shaping unit 16 can include a cylindrical optic, which compresses the illumination radiation BS in a direction perpendicular to the second optical axis A2. Downstream of the beam shaping unit 16 is a scanner 17, by which the shaped illumination radiation BS can be deflected in two directions perpendicular to the second optical axis A2 (so-called XY scanner). The illumination radiation BS deflected by the scanner 17 reaches the illumination objective 2 and is directed by it into the sample 5.

[0062] The control unit 13 is provided for controlling the drives 14 and 23, the sample stage 11, the laser module 15, the beam shaping 16, the scanner 17 and / or the detector 18, and is connected to the elements to be controlled in a manner suitable for data transmission (indicated).

[0063] In further versions, the control unit 13 is additionally configured for the acquisition, storage, and / or evaluation of measured values. The control unit 13 can be used to control other elements and units of the microscope 1 and / or to receive and evaluate measured values ​​from them.

[0064] Based on the foregoing exemplary embodiments and illustrations, a technical implementation of a lens 3 according to the invention and its use will be explained below by way of example.

[0065] The detection angle of objective 3 is 25°, while the illumination angle (see above) is 65°. Objective 3 is specifically designed for the average refractive index of 1.36 typical for organoids. For this purpose, pure ethanol, which has a refractive index of 1.3608 and an Abbe number of 54.72, can be introduced as the immersion medium via an immersion medium feeder (neither of which are shown) into a space between sample carrier 7 and objective 3, or optionally between sample carrier 7 and a specially designed optical lens (immersion end lens) in front of objective 3.

[0066] The object-side image field has a diameter of 1 mm and, in conjunction with a tube lens (not shown), is imaged onto the detector 18 with a lateral magnification of 10.69. The maximum numerical aperture in conjunction with the largest aperture diaphragm 10 is 0.6.

[0067] The focusing of the objective 3 is achieved by moving the sample stage 11, but can also be accomplished by varying the air gap between the immersion end lens and the objective 3. The imaging errors caused by the oblique passage of the detection radiation beam DS through the base of the sample carrier 7 are compensated by the correction element 12, which in this example is positioned 1.28 mm in front of the plane of the pupil P (see Fig. 1) of the objective 3, and which has a flat entrance surface and a freeform exit surface as specified by the coefficients below.

[0068] The lens 3 is chromatically corrected for a wavelength range of at least 436 to 656 nm, with the imaging performance also decreasing only slightly in the remaining range between 400 nm and 750 nm.

[0069] The freeform surface of correction element 12 is developed in explicit form z(x,y) according to the equation given above. The polynomial coefficients are listed below, where, for example, the designation "X3Y4" indicates the polynomial coefficient for m = 3, n = 4. The normalization radius is 1.0.

[0070] The polynomial coefficients are:

[0071] Y: 8.3275E-04 X2: -5.9767E-04 Y2: -6.1226E-04

[0072] X2Y: 1.5153E-06 Y3: 1.7574E-06 X4: 7.3129E-06

[0073] X2Y2: 1.4597E-05 Y4: 7.2783E-06 X4Y: 2.7975E-09

[0074] X2Y3: 6.4280E-09 Y5: 3.8148E-09 X6: -3.4674E-09

[0075] X4Y2: -1.0789E-08 X2Y4: -1.1006E-08 Y6: -3.7554E-09

[0076] X6Y: 8.2067E-12 X4Y3: 2.5069E-11 X2Y5: 2.9657E-11

[0077] Y7: 9.1097E-12 X8: -5.6105E-11 X6Y2: -2.2170E-10

[0078] X4Y4: -3.3191E-10 X2Y6: -2.2065E-10 Y8: -5.4960E-11.

[0079] In order to illustrate corrections achievable with a lens 3 according to the invention, the following field points Fl to F13 are selected and evaluated as examples for an object-side image field with a diameter of 1.0 mm:

[0080] Fl: x = 0.00 mm y = 0.00 mm

[0081] F2: x = 0.00 mm y = -0.50 mm

[0082] F3: x = 0.00 mm y = -0.33 mm

[0083] F4: x = 0.00 mm y = 0.33 mm

[0084] F5: x = 0.00 mm y = 0.50 mm

[0085] F6: x = -0.50 mm y = 0.00 mm

[0086] F7: x = -0.33 mm y = 0.00 mm

[0087] F8: x = 0.33 mm y = 0.00 mm

[0088] F9: x = 0.50 mm y = 0.00 mm

[0089] F10: x = -0.28 mm y = -0.28 mm

[0090] Fill: x = 0.28 mm y = -0.28 mm

[0091] F12: x = -0.28 mm y = 0.28 mm

[0092] F13: x = 0.28mm y = 0.28mm .

[0093] For the respective assumed mean parameter values ​​of refractive index (n=l,3608) and thickness of the base of the sample carrier 7 (d=0.17 mm), the objective 3 provides excellent diffraction-limited imaging, as shown by polychromatic Strehl values ​​> 95 for all field points Fl to F13 (see Fig. 3).

[0094] Since no adaptive correction for variations in the thickness of the sample carrier 7 and the sample refractive index is provided, the image quality can decrease towards the edges of the respective parameter ranges (i.e., sample carrier 7 thicknesses of 0.15 mm and 0.19 mm, respectively, and sample refractive indices of 1.34 and 1.38, respectively). In these cases, diffraction-limited imaging is no longer possible. The resolving power can be increased by selecting a smaller aperture-limiting aperture, resulting in a smaller numerical aperture of the objective 3 and thus of the imaging beam path, since the resulting image aberrations also decrease with a smaller numerical aperture. A slight reduction in light-gathering efficiency is accepted in this process.

[0095] Table 1 below lists the characteristic values ​​of optical elements of the exemplary embodiment of a lens 3 according to the invention.

[0096] Object Type Refractive Index Abbe Number Object Type Refractive Index Abbe Number

[0097] 1 sphere 1,361 54,720 16 sphere

[0098] 2 sphere 1.526 55.000 17 sphere 1.439 94.946

[0099] 3 Sphere 1.361 54.720 18 Sphere 1.638 42.410

[0100] 4 Sphere 1,361 54,720 19 Sphere 1,592 68,370

[0101] 5 sphere 1,361 54,720 20 sphere

[0102] 6 sphere 1.677 49.829 21 sphere 1.738 32.261

[0103] 7 Sphere 22 xy-polynomial

[0104] 8 sphere 1.893 20.362 aperture 23

[0105] 9 sphere 24 sphere

[0106] 10 Sphere 1,439 94,946 25 Sphere 1,580 53,866

[0107] 11 sphere 26 sphere

[0108] 12 Sphere 1.702 41.239 TI Sphere 1.517 64.167

[0109] 13 sphere 28 sphere

[0110] 14 Sphere 1,487 84,468 Image

[0111] 15 Sphere 1.638 42.410 Table 2 gives Y-radii and thicknesses of the optical elements of the exemplary embodiment of a lens 3 according to the invention. Object Type Y-Radius Thickness Object Type Y-Radius Thickness

[0112] 1 sphere infinity 0.0 16 sphere 12.78 2.7

[0113] 2 sphere infinity 1.7 17 sphere 20.42 8.5

[0114] 3 sphere infinity 0.0 18 sphere -14.55 1.6

[0115] 4 sphere infinity 3.0 19 sphere 19.56 5.7

[0116] 5 sphere -3.50 0.0 20 sphere -50.85 3.0

[0117] 6 sphere -3.50 2.9 21 sphere infinity 2.5

[0118] 7 sphere -6.44 1.9 22 xy-polynomial infinity 1.3

[0119] 8 sphere -4.47 5.3 aperture 23 infinity 22.9

[0120] 9 sphere -9.31 0.3 24 sphere infinity 126.5

[0121] 10 sphere -32.92 4.8 25 sphere 189.42 10.9

[0122] 11 Sphere -14.01 1.2 26 Sphere -189.42 60.0

[0123] 12 Sphere 40.63 5.3 I Sphere infinity 80.0

[0124] 13 sphere -74.06 0.1 28 sphere infinity 48.2

[0125] 14 Sphere 15.91 7.7 Image infinity 0.0

[0126] 15 Sphere 217.52 1.7 2.7

[0127] Based on the above embodiment, residual wavefront errors for various wavelengths (435.8 nm; 486.1 nm; 546.1 nm) are shown in the table in Figure 3.

[0128] The wavelengths 587.6 nm and 656.3 nm are listed. A wavefront analysis with RMS values ​​and the resulting Strehl ratios for field points Fl to F13 of lens 3 are given with respect to the wavelengths 435.8 nm, 486.1 nm, 546.1 nm, 587.6 nm, and 656.3 nm. Columns 6 and 7 show the RMS value and the resulting Strehl ratio in the optimal (refocused) plane for the respective field point, while columns 9 and 10 show the corresponding values ​​for the best common focal plane. The good field flatness of lens 3 is evident from the fact that no differences are discernible down to the third decimal place. The Strehl values, which are consistently above 0.95, demonstrate the almost perfectly diffraction-limited imaging quality of the lens 3 over the entire wavelength range – for a mean sample carrier thickness d = 0.17 mm and a mean sample refractive index n = 1.3608.The determined values ​​prove that the correction element 12 according to the invention, optionally in conjunction with a suitable aperture diaphragm 10, almost completely compensates aberrations and enables practically diffraction-limited imaging in all positions.

[0129] For the field points Fl to F5 (Fig. 4), for the field points F6 to F10 (Fig. 5) and for the field points Fll to F13 (Fig. 6) the residual wavefront errors with respect to the wavelengths 435.8 nm;

[0130] 486.1 nm; 546.1 nm; 587.6 nm and 656.3 nm shown in units of the reference wavelength 546.1 nm.

[0131] Reference sign

[0132] 1 microscope

[0133] 2 Lighting lenses

[0134] 3 Detection lens

[0135] 4 Sample level 5 Sample

[0136] 6 light leaf

[0137] 7 Sample carriers

[0138] 8 Medium

[0139] 9.1 first lens group

[0140] 9.11 Front lens

[0141] 9.12 optical lens

[0142] 9.2 second lens group

[0143] 9.21 optical lens

[0144] 10 Aperture stop

[0145] 11 Sample table

[0146] 12 first correction element

[0147] 13 Control unit

[0148] 14 Drive

[0149] 15 laser modules

[0150] 16 Beam shaping

[0151] 17 XY scanners

[0152] 18 Detector

[0153] 19 Magazine

[0154] 20 aperture changers, changers

[0155] 21 optical arrangement

[0156] 22. Breakthrough, hole, recess

[0157] 23 Drive (of the aperture changer 20)

[0158] First optical axis

[0159] A2 second optical axis

[0160] P Pupil, pupil plane

[0161] BE Image plane

[0162] BS lighting radiation

[0163] DS detection radiation

[0164] N surface normal (of the sample plane 4)

Claims

Patent claims 1. Objective (3), in particular a detection objective for a microscope (1), comprising a plurality of optical elements arranged along an optical axis (Al) in the beam path of the objective (3), characterized in that a pupil (AP) is formed outside a sequence of light transmission surfaces of the optical elements along the optical axis (Al) and following them in the direction of light in the beam path.

2. Lens (3) according to claim 1, characterized in that a replaceable first correction element (12) is provided for reducing aberrations which can be arranged manually or mechanically in or near the pupil (AP).

3. Lens (3) according to claim 2, characterized in that at least one light transmission surface of the first correction element (12) is designed as a freeform surface.

4. Lens (3) according to claim 3, characterized in that the freeform surface is symmetrical about a plane of symmetry spanned by the optical axis (Al) and a normal to the optical axis (Al).

5. Objective (3) according to one of claims 3 and 4, characterized in that the freeform surface is defined by an equation of the form is described where x, y and z denote the three Cartesian coordinates of a point lying on the freeform surface, and to describe the freeform surface in a direction orthogonal to the plane of symmetry only even powers not equal to zero and parallel to the plane of symmetry both even and odd powers not equal to zero are used.

6. Lens (3) according to one of the preceding claims, characterized in that the lens (3) has a numerical aperture less than or equal to 0.

6.

7. Lens (3) according to one of claims 2 to 5, characterized in that in addition to the first correction element (12) further correction elements are present.

8. Lens (3) according to one of the preceding claims, characterized in that a manually or mechanically interchangeable aperture diaphragm 10 is provided in or near the pupil (AP).

9. Lens (3) according to claim 8, characterized in that a changer (20) with a number of different aperture diaphragms 10 is provided, by means of which a selected aperture diaphragm 10 can be placed in the beam path.

10. Lens (3) according to one of the preceding claims, characterized in that an image field of > 0.8 mm, advantageously of > 0.9 mm, particularly preferably of > 1.0 mm can be captured on the object side.

11. Optical arrangement (21) comprising a lens (3) according to claim 2 or any one of claims 3 to 10 referring back to claim 2, and a sample table (11) with a support surface for holding a sample (5), wherein the support surface defines a sample plane (4), and wherein the optical axis (Al) of the lens (3) is directed into the sample plane (4) at an angle greater than 0°, measured between the optical axis (1) and a surface normal (N) standing on the sample plane (4).

12. Optical arrangement (21) according to claim 11, characterized in that the freeform surface of the correction element (12) is symmetrically designed, wherein a meridional plane serves as the plane of symmetry, which is spanned by the optical axis (Al) of the lens (3) and a surface normal (N) of the sample plane (4).

13. Microscope (1), in particular a light-sheet microscope, with an objective (3) according to one of claims 1 to 10 or with an optical arrangement (21) according to one of claims 11 and 12.

14. Use of a microscope (1) according to claim 13 for area-wise imaging of organoids.

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