OPTICAL SYSTEM FOR A MICROSCOPE

Description

Technical field

[0001] The present invention relates to an optical system for a microscope for imaging an object. Furthermore, the invention relates to a microscope having such an optical system and a method for imaging an object using a microscope. Background of the invention

[0002] In the field of microscopy, optical systems are known that enable volumetric imaging of a sample. In contrast to conventional microscopic imaging, in which the sample under investigation is usually imaged in a plane perpendicular to the optical axis, this type of volumetric imaging enables the imaging of a sample region extending deep into the sample. For this purpose, microscopes that illuminate only a thin layer of the sample with a planar illumination light distribution, also known as a light sheet or light disk, are preferred. In this specific application, the light sheet is often positioned at an angle to the optical axis.

[0003] Microscopes designed for volumetric imaging typically feature a telescope system that projects the target area of ​​the sample from the object space into the image space. To enable largely aberration-free imaging, the magnification of the telescope system should be selected to correspond to the ratio of the refractive indices of the optical media arranged in the object and image spaces. These refractive indices typically differ from one another. Particularly in the microscopic examination of biological samples, the requirement for aberration-free volumetric imaging—namely, adapting the magnification of the telescope system to the ratio of the object-side refractive index to the image-side refractive index—is difficult to meet.For example, in a biological sample, its refractive index often varies significantly with the imaging depth within the sample, which leads to a corresponding variation of the aforementioned refractive index ratio to which the magnification of the telescope system must be adjusted.

[0004] DE 10 2016 119 268 B3 discloses transport optics for a light sheet microscope. The transport optics comprise an afocal system configured to adapt the refractive index ratio between a sample space and an intermediate image space. For the prior art, reference is further made to documents WO 2017 / 0 150 177, US 2016 / 0114554 A1, and DE 10 2008 026 774 A1.

[0005] Sample-related refractive index mismatches are particularly detrimental in common microscopic applications, as the optical systems used there often have a high numerical aperture. The spherical aberrations associated with a refractive index mismatch are therefore correspondingly large.

[0006] The object of the invention is to provide an optical system for a microscope that enables largely aberration-free object imaging, regardless of any differences in the refractive indices in the object and image space. Furthermore, the object of the invention is to provide a microscope with an optical system and a method for imaging an object using such a microscope.

[0007] The invention solves this problem by the subject matter of the independent claims. Advantageous further developments emerge from the dependent claims and the following description.

[0008] An optical system intended for a microscope is proposed, comprising a telescope system with an optical correction unit which is adjustable for correcting a spherical aberration, and with zoom optics which are adjustable for adapting the magnification of the telescope system to the ratio of two refractive indices, one of which is assigned to the object side and the other to the image side, within a predetermined magnification range, wherein the telescope system is designed to be telecentric with respect to both the object side and the image side over the entire magnification range due to the zoom optics contained therein.

[0009] In this embodiment, the zoom optics enable variable zoom adjustment depending on the refractive index mismatch present in the specific microscopic application. In particular, the zoom optics allow the magnification to be adjusted to a value that is essentially equal to the ratio between the refractive index in object space and the refractive index in image space.

[0010] As part of the telescope system, the zoom optics ensure that the telescope system as a whole is telecentric on both sides, i.e., with respect to both the object space and the image space. To this end, the zoom optics form a so-called pupil-controlled zoom system, which enables volumetric imaging. An example of such a pupil-controlled zoom system is described in H. Gross, Handbook of Optical Systems, Vol. 4, ISBN 978-3-527-40380-6, Wiley-VCH Weinheim 2008, pp. 478-482.

[0011] In the present embodiment, the afocal telescope system further includes an optical correction unit, which serves to correct spherical aberrations, particularly those occurring at high numerical apertures. The zoom optics and the aforementioned correction unit thus provide two degrees of freedom for adjusting the telescope system, which, depending on the application, can be used individually or together for largely aberration-free sample imaging. Preferably, both the zoom optics and the correction unit can be adjusted motorically.

[0012] The telescope system includes a first objective lens facing the object side and a second objective lens facing the image side. Furthermore, the zoom optics are preferably configured to image the focal plane of the first objective lens facing the image side onto the focal plane of the second objective lens facing the object side across the entire magnification range. This imaging property characterizes the zoom optics as a pupil-controlled zoom system, enabling largely error-free volume imaging even with variable refractive index mismatches. The ability of the zoom optics to act as a pupil-controlled system can also be understood as meaning that the zoom optics images the pupil of the object-side objective lens onto the pupil of the image-side objective lens across the entire magnification or zoom range, with the aforementioned objectives being designed as telecentric systems.

[0013] The correction unit is contained in the first objective or the second objective. In this embodiment, the correction unit is formed, for example, from an objective lens group that can be adjusted along the optical axis of the objective to correct the spherical aberration. Since the two objectives have the same aperture angles when the magnification of the telescope system is adapted to the ratio of the refractive indices in the object and image space, it is freely selectable which of the two objectives is to be equipped with the correction unit. This enables a particularly flexible optical design.

[0014] In a preferred embodiment, the magnification range of the telescope system corresponds to a range in which the ratio of the two refractive indices lies between 1.0 and 1.6. If the zoom optics are designed to realize such a magnification range, sample-induced refractive index mismatches can be compensated within wide limits.

[0015] In a preferred embodiment, the telescope system includes a Kepler telescope system arranged between the first objective and the second objective, which contains the zoom optics. With such a Kepler telescope system, the afocal telescope system can be implemented in a comparatively simple manner.

[0016] Preferably, the Kepler telescope system contains two tube lens units, one of which forms the zoom optics. For example, the zoom optics is provided by the one of the two tube lens units located on the object side of an intermediate image plane within the Kepler telescope system.

[0017] In a further preferred embodiment, the zoom optics contain at least three lens units, which are preferably movable independently of one another along the optical axis of the telescope system to change the magnification. Each of the aforementioned lens units represents a degree of freedom for the desired pupil adjustment.

[0018] In a particularly preferred embodiment, the optical system comprises a focusing device configured to move the first objective along its optical axis relative to the object, thereby changing the distance of the first objective from the Kepler telescope system. Furthermore, the three lens units of the zoom optics are movable independently of one another along the optical axis such that, as the distance of the first objective from the Kepler telescope system changes, the focal plane of the first objective facing the image side is imaged onto the focal plane of the second objective facing the object side, while maintaining the magnification set by the zoom optics and the afocality of the telescope system.In this embodiment, a so-called objective focusing system is implemented, i.e., a focusing system in which the objective facing the object (but not, for example, a microscope stage supporting the object) is moved along the optical axis to achieve the desired focus. With this type of objective focusing system, the three lens units of the zoom optics offer three controllable degrees of freedom, which are determined by the positions of the three lens units along the optical axis, allowing the pupil adjustment to be used to freely adjust the pupil image within limits.This allows the position of the image of the aforementioned image-side focal plane and the position of an intermediate image in the Kepler telescope system to be adjusted during lens focusing, which changes the axial distance of the object-side lens from the Kepler telescope system and thus also the imaging of the image-side focal plane of this lens through the Kepler telescope system. This makes it possible, in particular, to maintain bilateral telecentricity even when using lens focusing, e.g., revolver focusing, by appropriately controlling the individual lens units of the zoom optics. In order to be able to adjust the three setting variables—namely, telecentricity, afocality, and magnification—independently of one another in this case, it is advantageous to control the three degrees of freedom determined by the axial positions of the three lens groups of the zoom optics independently of one another.

[0019] In a preferred embodiment, the optical system comprises a device configured to detect the refractive index associated with the object space, and a controller configured to adjust the optical correction unit and the zoom optics depending on the detected refractive index. The aforementioned detection device can, for example, be designed to measure the refractive index in situ and supply it to the controller as a control parameter. In a simplified embodiment, however, this device can also serve merely to enable the user to provide an input via which the refractive index is made available to the controller as a control parameter.

[0020] In a particularly preferred embodiment, the controller is designed to adjust the optical correction unit and the zoom optics in a coupled manner depending on the detected refractive index. Such a coupled adjustment means that the two originally available adjustment degrees of freedom, namely the correction adjustment and the zoom adjustment, are reduced to a single degree of freedom. This can be achieved, for example, by specifying a working plane onto which the telescope system is focused. By specifying a fixed working plane within the object, a fixed coupling between the zoom and correction adjustments is implemented in the telescope system, so that the value for the remaining single degree of freedom correlates only with the object-side refractive index, but not with the imaging depth within the sample.If the refractive index is known, an optimal value of the aforementioned degree of freedom can be determined.

[0021] In a further refined embodiment, a device is further provided which is designed to detect the distance of the telescope system from a reference plane which defines the position of the object relative to the telescope system. The controller is then preferably designed to additionally adjust at least the optical correction unit as a function of the detected distance. The surface of a cover glass, for example, can be used as the reference plane, based on which the distance between the object and the telescope system along its optical axis can be determined. In this alternative embodiment, the two degrees of freedom provided by the correction adjustment and the zoom adjustment are therefore no longer firmly coupled to one another, in contrast to the embodiment described above.Knowing the distance between the telescope system and the reference plane, and simultaneously knowing the object-side refractive index, both the spherical correction for the currently set object plane and the magnification ratio can be adjusted to the ratio between the object-side refractive index and the image-side refractive index. In this way, an optimal correction of the volume image can be achieved automatically for every position along the optical axis within the object.

[0022] In a further embodiment, the telescope system contains a light deflection device arranged within the telescope system at the location of an intermediate pupil. The intermediate image plane can be generated, for example, with the aid of another telescope system. The light deflection device can be used advantageously, particularly in a light-sheet microscopy application, e.g., for generating the light sheet itself and / or for adjusting it in order to scan the sample with the light sheet.

[0023] According to a further aspect, a microscope with an optical system of the type described above is provided.

[0024] Such a microscope can, for example, be designed as a light sheet microscope. In particular, the microscope is designed, for example, in the form of an OPM or SCAPE microscope suitable for volume imaging. OPM stands for "oblique plane microscope" and SCAPE for "swept confocally aligned planar excitation." These two microscope configurations are characterized by the fact that the sample is illuminated and imaged through the same objective. In these configurations, this results in the generated light sheet being oblique to the optical axis. Accordingly, in an OPM or SCAPE configuration, it is also ensured that the detection plane, i.e. the plane within the sample that is imaged onto the detector, is aligned obliquely to the optical axis.

[0025] According to a further aspect of the invention, a method for imaging an object using a microscope with a telescope system is provided, which includes an optical correction unit and a zoom lens. The method provides the following steps: correcting a spherical aberration by adjusting the optical correction unit and adapting the magnification of the telescope system within a predetermined magnification range to the ratio of two refractive indices, one of which is assigned to the object side and the other to the image side, by adjusting the zoom lens. The method further provides that the refractive index assigned to the object side is detected and the optical correction unit and the zoom lens are adjusted depending on this refractive index.

[0026] In a preferred embodiment, a working plane is specified on which the telescope system is focused. The optical correction unit and the zoom optics are coupled to each other and adjusted depending on the detected refractive index. In this embodiment, the two adjustment degrees of freedom, namely the adjustment of the correction unit and the adjustment of the zoom optics, are thus coupled to form a single degree of freedom, with this remaining degree of freedom correlating only with the object-side refractive index.

[0027] In a further embodiment, the distance of the telescope system from a reference plane is detected, which defines the position of the object relative to the telescope system. At least the optical correction unit is additionally adjusted depending on the detected distance. In this case, the two degrees of freedom of the correction and zoom adjustment are not rigidly coupled.

[0028] Embodiments of the invention are explained in more detail below with reference to the figures, in which: Fig. 1 is a schematic representation of a microscope having an optical system according to an embodiment; Fig. 2 is a schematic representation of a modified embodiment of the microscope according to Figure 1 ; Fig. 3 a schematic representation of a device suitable for use in the optical system according to Fig. 2 to determine a refractive index in the object space; Fig. 4 an object space of the microscope according to Figure 2 to illustrate how by means of the device according to Fig. 3 the refractive index is determined; Fig. 5 a by a position-sensitive detector of the device according to Figure 3 recorded intensity distribution for determining the refractive index; Fig. 6 the object space of the microscope according to Figure 2 to illustrate how by means of the device according to Fig. 3the distance of the optical system to a cover glass is determined; Fig. 7 a by the position-sensitive detector of the device according to Figure 3 detected intensity distribution for distance determination; Fig. 8 a lens diagram of the object-side objective of the optical system as an embodiment; Fig. 9 a schematic representation of the microscope containing an optical system according to a further embodiment; Fig. 10 a schematic representation of a modified embodiment of the microscope according to Figure 9; Fig. 11 is a flowchart showing an adjustment method as an embodiment in which an optical correction unit and a zoom optics are controlled in a coupled manner; Fig. 12 is a flowchart showing an adjustment method as a further embodiment in which the optical correction unit and the zoom optics are controlled independently of one another; and Fig. 13 is a schematic diagram illustrating how a working plane is defined in an OPM or SCAPE configuration.

[0029] The schematic representation according to Figure 1 shows an optical system 100 which is part of a Figure 1 microscope generally designated 102. In Figure 1 (As in the other figures) only those components of the microscope 102 are shown which are necessary for understanding the embodiment.

[0030] The optical system 100 according to Figure 1comprises a telescope system 104, which serves to image an object 106 lying in an object space into an image space, i.e., to generate an image 108 of the object 106. For this purpose, the telescope system 104 contains, starting from the object side towards the image side, a first objective 110 with a rear, i.e., image-side focal plane 112, a zoom optics 114, which in the present embodiment forms a first tube lens unit 116 and is formed from three lens units 118, 120, 122 movable along the optical axis O of the telescope system 104, an intermediate image plane 124, a second tube lens unit 126, and a second objective 128 with a rear, i.e., object-side focal plane 130 in the present configuration. The two tube lens units 114, 126 form a Kepler telescope system 132.The telescope system 104 further comprises an optical correction unit, which in the present embodiment is integrated into the object-side objective 110 and serves to correct a spherical aberration. An embodiment of the correction unit will be described later with reference to FIG. Figure 8 explained in more detail.

[0031] The zoom optics 114 have the function of adapting the magnification of the telescope system 104 within a predetermined magnification range to the ratio of two refractive indices, one of which is assigned to an optical medium in the object space and the other to an optical medium in the image space. The object-side refractive index can, for example, refer to an embedding medium in which the object 106 is embedded or to the object 106 itself. Particularly when the microscope 102 is used for volume imaging, it may be necessary to know the refractive index within the object as a function of the imaging depth for largely aberration-free imaging.

[0032] To set a desired magnification, in the present embodiment, one or more of the lens units 118, 120, 122 of the zoom optics 114 are moved along the optical axis O. Thus, a specific axial positioning of the lens units 118, 120, 122 corresponds to a specific zoom setting, which in turn effects the desired magnification of the telescope system 104.

[0033] The optical system 100 according to Figure 1 has a control 134 via which the correction unit integrated in the object-side lens 110 and the zoom optics 114 can be controlled in order to carry out the desired correction setting or the desired zoom setting.

[0034] In the present embodiment, the zoom optics 114 are designed as a pupil-regulated zoom system. Accordingly, the zoom optics 114 images the image-side focal plane 112 of the object-side objective 110 over the entire magnification range, ie, for all zoom settings, onto the object-side focal plane 130 of the image-side objective 128. As in Figure 1 As shown in dashed lines, this means that the zoom optics 114 images the pupil of the object-side objective 110 onto the pupil of the image-side objective 128 across the entire magnification or zoom range. The two objectives 110, 128 themselves are designed as telecentric systems. As a result, the telescope system 104 as a whole forms a two-sided system, i.e., a telecentric system with respect to both the object side and the image side.

[0035] In the present embodiment, the optical system 100 may further comprise a focusing device, which in the illustration according to Figure 1 is indicated purely schematically by a double arrow 136. The focusing device 136 can be controlled via the control 134 in such a way that it moves the object-side objective 110 as a whole along the optical axis O towards or away from the object 106 in order to focus on a desired plane within the object 106. This axial movement of the objective 110 changes the distance of its image-side focal plane 112 with respect to the Kepler telescope system 132 formed from the two tube lens units 114, 126. Thus, in the Figure 1 In the embodiment shown, a so-called lens focusing is realized.

[0036] In order to compensate for the aforementioned distance change during such lens focusing, the three lens units 118, 120, 122 of the zoom optics 114 can be controlled independently of one another. In this way, the pupil imaging can be controlled such that any desired magnification scale can be set within the predetermined magnification range while simultaneously maintaining the bilateral telecentricity and afocality of the telescope system 104.

[0037] In the present embodiment, the axial adjustment paths of the lens units 118, 120, 122 forming the zoom optics 114 are selected such that the range within which the magnification of the telescope system 104 can be varied corresponds to a range in which the ratio between the object-side refractive index and the image-side refractive index is between 1.0 and 1.6.

[0038] In the Figure 1In the exemplary embodiment shown, the adjustment of the correction unit integrated in the lens 110 and the adjustment of the zoom optics 114 are coupled to one another. Through this coupling, the two actually available degrees of freedom, namely the correction adjustment and the zoom adjustment, are reduced to a single degree of freedom, which in this case is controlled as a function of the object-side refractive index. Thus, in the exemplary embodiment according to Figure 1, the object plane onto which the telescope system 104 is focused is predetermined. This object plane defines a fixed working plane within the object 106, so that the imaging depth is fixed. The only control parameter that remains, based on which the correction of the spherical aberration and the zoom adjustment are carried out, is the refractive index assigned to the object space. The latter can be transmitted to the controller 134, for example, via a Figure 1input device not shown.

[0039] In a modified embodiment, the refractive index can also be measured in situ by the microscope 102 itself. Such a modification is described in Figure 2 shown.

[0040] The microscope 102 after Figure 2 additionally comprises a splitter mirror 238 and a detection device 240, which is designed to detect the refractive index in the object space in situ. The splitter mirror 238 and the detection device 240 are in Figure 3 presented in detail.

[0041] As in Figure 3As shown, the detection device 240 has a light source 342 that emits a measuring light beam 344 in the infrared wavelength range. The light source 342 is, for example, an LED that has a slit diaphragm 346 through which the measuring light beam 344 is directed onto an illumination optics 348. After passing through the illumination optics 348, the measuring light beam 344 falls on an aperture diaphragm 350 that is positioned centrally on the optical axis O1 of the illumination optics 348 and has an aperture 352 that is arranged off-center at a distance from the optical axis O1. The aperture 352 of the aperture diaphragm 350 limits the beam cross-section of the measuring light beam 344 such that only the Figure 3 the part of the measuring light beam 344 lying below the optical axis O1 of the illumination optics 348 passes the aperture stop 350 in the direction of a deflection prism 354.

[0042] The measuring light beam 344, limited in its beam cross-section, is reflected by the deflection prism 354 into a transport optics 356, which is formed by a focusing lens 358 movable along its optical axis O2, a stray light diaphragm 360, and another lens 362. After passing through the transport optics 356, the measuring light beam 344 falls on the beam splitter 238, which is designed as a dichroic beam splitter. The beam splitter 238 reflects light in the infrared wavelength range while transmitting light in the visible range. The measuring light beam 344 is reflected by the beam splitter 238 toward the objective 110. The measuring light beam 344 reflected by the beam splitter 238 travels with a parallel offset to the optical axis O of the objective 110.In this way, the measuring light beam 344 is guided into a partial area of ​​an entrance pupil 364 of the objective 110, which is laterally offset with respect to the optical O of the objective 110 and thus with respect to the center of the entrance pupil 364 (see also . Figure 4 ). The entrance pupil 364 of the objective 110 is thus illuminated decentrally, which leads to the measuring light beam 344 being directed into the object space at an angle α oblique to the optical axis O.

[0043] With reference to Figure 4 For the present embodiment, it should be assumed that a cover glass 424 is located in the object space of the microscope 102, which serves to Figure 4 not explicitly shown object. On the cover glass 424 there is an embedding medium 426 in which the object is embedded. In the object space 414 there is also an immersion medium 428 which in the illustration according to Figure 4borders on the objective 110 from above and on the cover glass 424 from below.

[0044] For simplicity, Figure 3 the embedding medium 426 and the immersion medium 428, which border the cover glass 424 from opposite sides in the object space, are omitted. The measuring light beam 344, which is directed into the object space at an oblique incidence, is, as described below with reference to Figure 4 explained in more detail, is reflected on the cover glass 424, whereby two reflected light beams are formed which are guided back into the objective 110 and which, in the schematic view, Figure 3 (in contrast to the detailed view after Figure 4 ) are represented in the form of a single light beam designated 366.

[0045] After passing through the lens 110, the two reflected light beams 366 fall onto the splitter mirror 238, which directs the reflected light beams 366 into the transport optics 356. After passing through the transport optics 356, the reflected light beams 366 fall onto the deflection prism 354, which reflects the reflected light beams 366 onto a detector optics 368. The detector optics 368 directs the reflected light beams 366 onto a spectral filter 370, which is only transparent to light in the infrared wavelength range and blocks stray light outside this wavelength range. The reflected light beams 366 transmitted through the spectral filter 370 finally fall onto a position-sensitive detector 372, which is capable of detecting the intensities of the reflected light beams 366 with spatial resolution.

[0046] In Figure 4 It is shown in more detail how, by reflection of the measuring light beam 344, the two (in Figure 4designated 366a, 366b) reflection light beams are generated, which in the present embodiment are used to determine the refractive index of the embedding medium 426. Accordingly, the measuring light beam 344 decentrally illuminating the entrance pupil 364 of the objective 110 is directed through the objective 110 at an angle α obliquely to the optical axis O onto the Figure 4 directed to the front surface of the cover glass 424, designated 464. Since the cover glass 424 and the immersion medium 428 bordering its front surface 464 have different refractive indices, the front surface 464 of the cover glass 424 and the adjacent immersion medium 28 form a first interface at which the incident measuring light beam 344 is partially reflected. The portion of the measuring light beam 344 reflected at this first interface generates the first reflected light beam 366a, which is guided back into the objective 110.

[0047] The other part 466 of the measuring light beam 344, which transmits the first interface, is refracted away from the optical axis O of the objective 110 upon entering the cover glass 424 and forms an angle β with it that is greater than the angle α. This transmitted part 466 of the measuring light beam 344 is partially reflected at a second interface defined by the rear surface 468 of the cover glass 424 and the adjacent embedding medium 426, which has a different refractive index than the cover glass 424. This second partial reflection of the measuring light beam 344 at the second interface generates the second reflected light beam 366b, which passes through the front surface 464 of the cover glass 424 and then returns to the objective 110.

[0048] As shown in the illustration Figure 4As illustrated, the oblique incidence of the measuring light beam 344 into the object space ensures that the reflection light beams 366a, 366b generated by the two partial reflections on the front surface 464 and the rear surface 468 of the cover glass 424 return to the objective 110 via different optical paths. In this way, the two reflection light beams 366a, 366b strike the position-sensitive detector 372 at different incidence points. In other words, the two measurement patterns generated on the front surface 464 and the rear surface 468 of the cover glass 424 in the form of slit images are imaged spatially separated from one another onto the position-sensitive detector 372, as shown in the diagram according to Figure 5 is illustrated.

[0049] Figure 5shows an exemplary intensity distribution V, which the two reflected light beams 366a, 366b jointly generate on the position-sensitive detector 372. The abscissa 570 of the diagram represents the point of incidence on the detector 372, and the ordinate 572 represents the intensity measured at the respective point of incidence. The intensity distribution V according to Figure 5 shows two peaks, of which the peak labeled P1 is associated with the first reflected light beam 366a and the peak labeled P2 with the second reflected light beam 366b. From the fact that peak P1 is higher and sharper than peak P2, it can be seen that in the example according to Figure 4the measuring light beam 344 is focused on the front surface 464 of the cover glass 424. This means that a focused image of the slit 346 of the light source 342 is generated on the front surface 464 of the cover glass 424, while a defocused image of the slit 346 is generated on the rear surface 468 of the cover glass 424. This corresponds to the illustration according to Figure 4 , as the first partial reflection takes place on the front surface 464 of the cover glass 424 at a point centered on the optical axis O of the objective 110. In contrast, the second partial reflection takes place on the rear surface 468 of the cover glass 424 at a point offset transversely to the optical axis O. The surfaces below the Figure 5 The peaks P1, P2 shown are each a measure of the intensity of the respective reflected light beam 366a, 366b.

[0050] In the present example, it is assumed that the numerical aperture of the measuring light beam 344 and the refractive indices of the immersion medium 428 and the cover glass 24 are known. Knowing these values, the refractive index of the embedding medium 426 can be calculated from the in situ determined ratio of the intensities of the peaks P1 and P2.

[0051] The refractive index, which is determined in situ by the detection device 240 as explained above, represents in the embodiment according to Figure 2represents a control parameter that is fed to the controller 134. Based on this control parameter, the controller 134, as described above, performs a coupled adjustment of the correction unit and the zoom optics 114. It goes without saying that the aforementioned control parameter is not limited to the refractive index of an embedding medium. In particular, the refractive index of the object itself can also be used as a control parameter.

[0052] The Figure 2The exemplary embodiment shown can be further modified in that the two available degrees of freedom, namely the correction setting and the zoom setting, are controlled independently of one another. In this case, a further control parameter is added, which corresponds, for example, to the distance of the telescope system 104 from a reference plane, wherein this reference plane defines the position of the objective 110 relative to the telescope system 104. In order to detect this additional control parameter in situ, the detection device 240 shown in Figures 2 and 3 can be operated in a manner which is exemplary in Figure 6 is illustrated.

[0053] In the example after Figure 6 In contrast to the situation in Figure 4only the reflection of the measuring light beam 344 at the front surface 464 of the cover glass 424 is taken into account. Accordingly, only that part of the measuring light beam 344 which is reflected at the interface formed by the front surface 464 of the cover glass 424 and the adjacent immersion medium 428 is evaluated.

[0054] In Figure 7 the corresponding intensity distribution V is shown, which the reflected light beam 366a generates on the position-sensitive detector 372. The intensity distribution V shows a peak P, whose position X, which is determined on the position-sensitive detector 372 with respect to a reference position X ref, is a measure of the Figure 6 is the distance z shown, which the front surface 464 of the cover glass 424 has along the optical axis O from the objective 110. The front surface 464 of the cover glass 424 forms the above-mentioned reference plane. Based on the position X, the distance z can thus be determined in situ.

[0055] In this embodiment, the two degrees of freedom, i.e., the correction setting and the zoom setting, are no longer rigidly coupled to one another. Because the detection device 240 detects both the distance of the telescope system 304 from the reference plane and the object-side refractive index, an optimal correction of the volume image can be achieved automatically for each position along the optical axis O within the object 106.

[0056] Figure 8 shows an embodiment of the lens 110 in which the correction unit intended for correcting the spherical aberration is integrated.

[0057] In the embodiment according to Figure 8The objective lens 110 comprises a first lens group 802 of positive refractive power, a second lens group 804 of positive refractive power, a third lens group 806 of negative refractive power, and a fourth lens group 808 of positive refractive power, arranged in this order from the object side. The second lens group 804 is adjustable along the optical axis O via the controller 134 to correct spherical aberration. In contrast, the first lens group 802, the third lens group 806, and the fourth lens group 808 are stationary.

[0058] The first lens group 802 is formed from a first lens 810 with negative refractive power and a second lens 812 with positive refractive power. The axially movable lens group 804 comprises a third lens 814 with positive refractive power, a fourth lens 816 with negative refractive power, and a fifth lens 818 with positive refractive power. The lenses 814, 816, and 818 form a cemented element. The third lens group 806 comprises a sixth lens 820 with positive refractive power and a seventh lens 822 with negative refractive power. The lenses 820, 822 also form a cemented element. Finally, the fourth lens group 808 is formed from an eighth lens 824 with negative refractive power and a ninth lens 826 with positive refractive power.

[0059] Figure 9shows a further embodiment of the optical system 100, in which the telescope system 104 has a further Kepler telescope system. The latter comprises, from the object side to the image side, a first eyepiece system 904, a light deflection device 906, and a second eyepiece system 908. The light deflection device 906 is formed, for example, from a raster mirror that can be rotated about two mutually perpendicular axes, as in Figure 9 indicated by the two arrows. The light deflection device is arranged at the location of an intermediate pupil. In the telescope system 104 according to Figure 9 Furthermore, a further intermediate image plane 910 is provided.

[0060] Apart from the additional Kepler telescope system, the design according to Figure 9 the in Figure 1 In particular, the embodiment according to Figure 9designed for a coupled adjustment of the correction unit and the zoom optics 114.

[0061] Figure 10 shows a further embodiment in which, in contrast to the one in Figure 9 In the configuration shown, the two degrees of freedom, namely the correction setting and the zoom setting, are controlled independently of each other. Accordingly, the embodiment according to Figure 10 also via the detection device 240 (according to the Figure 2 illustrated embodiment).

[0062] Figure 11 shows a flowchart illustrating an adjustment method in which the optical correction unit, ie the second lens group 804 of the objective 110 (cf. Figure 8 ), and the zoom optics 114 are controlled in a coupled manner.

[0063] First, in step S2, the refractive index in the object space is measured. In the example according to Figure 11The refractive index of the object itself should be determined.

[0064] Depending on the refractive index detected in step S2, the coupled control of the lens group 804 of the objective 110 and the zoom optics 114 then takes place in step S4. The setting made in step S4 is based on the specification of a fixed working plane on which the telescope system 104 is focused. Only this specification of a predefined working plane enables the implementation of a fixed coupling between the zoom and correction settings, whereby the only remaining degree of freedom correlates only with the refractive index, but not with the imaging depth within the object.

[0065] Finally, in step S6, the image acquisition is carried out based on the zoom and correction settings made in step S4.

[0066] Figure 12shows a flow chart to illustrate an adjustment method in which the optical correction unit, ie in the present embodiment the second lens group 804 of the lens 110, and the zoom optics 114 are controlled independently of one another.

[0067] In step S2, the object-side refractive index is again measured using the detection device 240. Subsequently, in step S4-1, the magnification is adjusted by controlling the zoom optics 114 depending on the refractive index detected in S2.

[0068] In contrast to the Figure 11 In the embodiment shown, the method according to Figure 12 Additionally, the distance between the object 106 and the objective 110 is determined using the detection device 240. This is done, for example, with reference to a special reference plane defined by the cover glass 424, as described above with reference to the Figure 6 and 7has been explained. In step S4-3, the correction adjustment is then carried out by moving the optical correction unit along the optical axis O of the lens 110 as a function of this distance.

[0069] Based on the settings made in steps S4-1 and S4-3, the image is finally captured in step S6.

[0070] In the example after Figure 12It is assumed that the adjustment of the zoom optics 114 can be made independently of the imaging depth, i.e., independently of the distance between the lens 110 and the object 106. Accordingly, in the magnification adjustment made in step S4-1, only the refractive index detected in step S2 is taken into account, but not the distance detected in step S4-2. However, it goes without saying that the distance detected in step S4-2 can also be taken into account when adjusting the magnification in step S4-1, if this should be advantageous.

[0071] Figure 13 Finally, a schematic representation shows how a working plane can be defined in the case of a coupled correction and zoom setting when the microscope 100 is operated for volume imaging, for example, in an OPM or SCAPE configuration.

[0072] As in Figure 6 is in the example after Figure 13assumed that the objective 110 is aligned with its optical axis O perpendicular to the cover glass 464. In this case, Figure 13 below the cover glass 464 the immersion medium 428 and above the cover glass 464 the embedding medium 426, into which the Figure 13 not explicitly represented object is embedded.

[0073] In the configuration according to Figure 13 The aforementioned working plane is defined by the plane within the object 106 on which the lens 110 is focused. This focal plane is in Figure 13 designated F and is perpendicular to the optical axis O of the lens 110.

[0074] In an OPM or SCAPE configuration, the lens 110 is used not only to image the object 106, but also to illuminate it. The lens 110 generates a light sheet that is oriented obliquely to the optical axis O of the lens 110. This inclination of the light sheet is compensated in this special configuration by correspondingly inclining a detection plane D relative to the optical axis O of the lens 110. This detection plane D defines the plane within the object 106 that is imaged onto an image sensor used for image generation.

[0075] The projection of the detection plane D onto the optical axis O defines the imaging depth of the volume imaging, ie the extent of the imaged sample volume along the optical axis O. The position of the working plane defined by the focal plane F of the objective 110 can now be suitably selected with respect to the detection plane D. In the example according to Figure 13 The focal plane F of the objective 110 is positioned relative to the detection plane D such that it lies approximately centrally along the optical axis O within the imaging depth defined by the detection plane D. It goes without saying, however, that this definition is only intended as an example. Taking into account any absorption or scattering effects, which naturally increase with the imaging depth, the working plane can also be defined differently relative to the detection plane D.

[0076] It goes without saying that the embodiments described above are only examples. For example, the Figure 3 The detection device 240 shown represents merely a specific example of how the refractive index and / or the distance of the telescope system 104 from a reference plane can be determined. However, the determination of the refractive index or the distance should in no way be limited to this example.

[0077] Although some aspects have been described in the context of an apparatus, it is to be understood that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a function of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block, element, or feature of a corresponding apparatus. Some or all of the method steps may be performed by (or using) a hardware device, such as a processor, a microprocessor, a programmable computer, or an electronic circuit. In some embodiments, one or more of the key method steps may be performed by such a device.

[0078] Depending on specific implementation requirements, embodiments of the invention may be implemented in hardware or software. The implementation may be performed using a non-volatile storage medium, such as a digital storage medium, such as a floppy disk, a DVD, a Blu-ray disc, a CD, a ROM, a PROM and EPROM, an EEPROM, or a FLASH memory, on which electronically readable control signals are stored that interact (or can interact) with a programmable computer system to perform the respective method. Therefore, the digital storage medium may be computer-readable.

[0079] Some embodiments according to the invention comprise a data carrier with electronically readable control signals that can interact with a programmable computer system so that one of the methods described herein is carried out.

[0080] In general, embodiments of the present invention can be implemented as a computer program product with program code, wherein the program code is effective for executing one of the methods when the computer program product is run on a computer. The program code can, for example, be stored on a machine-readable medium.

[0081] Further embodiments include the computer program for carrying out one of the methods described herein, which is stored on a machine-readable carrier.

[0082] In other words, one embodiment of the present invention is therefore a computer program having program code for carrying out one of the methods described herein when the computer program is running on a computer.

[0083] A further embodiment of the present invention is therefore a storage medium (or a data carrier or a computer-readable medium) comprising a computer program stored thereon for performing one of the methods described herein when executed by a processor. The data carrier, the digital storage medium, or the recorded medium is typically tangible and / or non-transitory. A further embodiment of the present invention is an apparatus as described herein, comprising a processor and the storage medium.

[0084] A further embodiment of the invention is therefore a data stream or signal sequence representing the computer program for carrying out one of the methods described herein. The data stream or signal sequence can, for example, be configured to be transmitted via a data communication connection, for example, via the Internet.

[0085] Another embodiment comprises a processing means, for example a computer or a programmable logic device, configured or adapted to carry out any of the methods described herein.

[0086] A further embodiment comprises a computer on which the computer program for carrying out one of the methods described herein is installed.

[0087] A further embodiment according to the invention comprises a device or system configured to transmit (e.g., electronically or optically) a computer program for performing one of the methods described herein to a recipient. The recipient may, for example, be a computer, a mobile device, a storage device, or the like. The device or system may, for example, comprise a file server for transmitting the computer program to the recipient.

[0088] In some embodiments, a programmable logic device (e.g., a field-programmable gate array, FPGA) may be used to perform some or all of the functionality of the methods described herein. In some embodiments, a field-programmable gate array may cooperate with a microprocessor to perform any of the methods described herein. In general, the methods are preferably performed by any hardware device. List of reference symbols

[0089] 100Optical system 102Microscope 104Telescope system 106Object 108Image 110Objective 112Focal plane 114Zoom optics 116Tube lens unit 118Lens unit 120Lens unit 122Lens unit 124Intermediate image plane 126Tube lens unit 128Objective 130Focal plane 132Kepler telescope system 134Control 136Focusing device 238Splitter mirror 240Detecting device 342Light source 344Measuring light beam 346Slit diaphragm 348Illumination optics 350Aperture diaphragm 352Aperture opening 354Deflection prism 356Transport optics 358Focusing lens 360Lens hood 362Lens 364Entrance pupil 366Reflection light beam 368Detector optics 370Spectral filter 372Detector 424Cover glass 426Embedding medium 428Immersion medium 464Front surface 466Part of the measuring light beam 468Rear surface 570Abscissa 572Ordinate 802Lens group 804Lens group 806Lens group 808Lens group 810Lens 812Lens 814Lens 816Lens 818Lens 820Lens 822Lens 824Lens 826Lens 904Eyepiece system 906Light deflection device 908Eyepiece system 910Intermediate image plane OpticalAxis O1optical axis O2optical axis FFocus plane DDetectional plane αAngle βAngle

Claims

1. An optical system (100) for a microscope (102) for imaging an object (106), comprising: a first objective (110) facing the object side and a second objective (128) facing the image side; a telescope system (104) arranged between the first objective (110) and the second objective (128), with zoom optics (114), which are adjustable, within a predetermined magnification range, to adapt the magnification of the telescope system (104) to the ratio of two refractive indices, one of which is assigned to the object side and the other to the image side, wherein the telescope system (104) is designed to be telecentric over the entire magnification range both with respect to the object side and with respect to the image side due to the zoom optics (114) contained therein, and wherein an optical correction unit (804) is contained in the first objective (110) or in the second objective (128), which optical correction unit is adjustable for correcting a spherical aberration.

2. The optical system (100) according to claim 1, in which the zoom optics (114) are designed to image the focal plane (112) of the first objective (110) facing the image side onto the focal plane (130) of the second objective (128) facing the object side over the entire magnification range.

3. The optical system (100) according to any one of the preceding claims, in which the magnification range of the telescope system (104) corresponds to a range in which the ratio of the two refractive indices is between 1.0 and 1.6.

4. The optical system (100) according to any one of the preceding claims, in which the telescope system (104) contains a Kepler telescope system (132) which is arranged between the first objective (110) and the second objective (128) and in which the zoom optics (114) are contained.

5. The optical system (100) according to claim 4, in which the Kepler telescope system (132) contains two tube lens units (114,126), one of which forms the zoom optics (114).

6. The optical system (100) according to any one of the preceding claims, in which the zoom optics (114) contain at least three lens units (118, 120, 122) which, in order to change the magnification of the telescope system (104), are movable along optical axis (O) thereof.

7. The optical system (100) according to claim 6, further comprising a focusing device (136) which is designed to move the first objective (110) along its optical axis (O) relative to the object (106), whereby the distance of the first objective (110) from the Kepler telescope system (132) changes, wherein the three lens units (118, 120, 122) of the zoom optics (114) are movable along the optical axis (O) independently of one another in such manner that when the distance of the first objective (110) from the Kepler telescope system (132) changes, the focal plane (112) of the first objective (110) facing the image side is imaged onto the focal plane (130) of the second objective (128) facing the object side, while maintaining the magnification set by the zoom optics (114) and the afocality of the telescope system (104).

8. The optical system (100) according to any one of the preceding claims, further comprising a device (240) which is designed to detect the refractive index, associated with the object space, and a controller (134) which is designed to adjust the optical correction unit (804) and the zoom optics (114) as a function of the detected refractive index.

9. The optical system (100) according to claim 8, in which the controller (134) is designed to adjust the optical correction unit (804) and the zoom optics (114) in a manner coupled to one another as a function of the detected refractive index.

10. The optical system (100) according to claim 8, further comprising a device (240) designed to detect the distance (z) of the telescope system (104) from a reference plane (464), which defines the position of the object (106) relative to the telescope system (104), wherein the controller (134) is designed to additionally adjust at least the optical correction unit (804) as a function of the detected distance (z).

11. A microscope (102) with an optical system (100) according to any one of the preceding claims.

12. A method for imaging an object (106) using a microscope (102) according to claim 11, with the following steps: correcting a spherical aberration by adjusting the optical correction unit (804), and adjusting the magnification of the telescope system (104) within a predetermined magnification range to the ratio of two refractive indices, one of which is assigned to the object side and the other to the image side, by adjusting the zoom optics (114), wherein the refractive index, associated with the object side, is detected and the optical correction unit (804) and the zoom optics (114) are adjusted as a function of this refractive index.

13. The method according to claim 12, in which a working plane (F) is predefined onto which the telescope system (104) is focused, and the optical correction unit (804) and the zoom optics (114) are adjusted in a manner coupled to one another as a function of the detected refractive index.

14. The method according to claim 12, in which the distance (z) of the telescope system (104) from a reference plane (464) is detected, which defines the position of the object (106) relative to the telescope system (104), and at least the optical correction unit (804) is additionally adjusted as a function of the detected distance (z).