MICROSCOPIC TRANSMITTED LIGHT CONTRASTING METHOD

Description

[0001] The invention relates to a microscopic transmitted light contrasting method and a device for carrying out a microscopic transmitted light contrasting method.

[0002] In the field of light microscopy, various phase-contrast techniques are known that aim to improve image contrast, particularly in the imaging of biological samples. The most well-known such techniques are Zernike phase contrast and Nomarski differential interference contrast, which are also available in conventional transmitted-light microscopes.

[0003] The classic Zernike phase-contrast method involves overlapping a light ring on the illumination side with a phase ring located in the objective pupil. The necessity of such overlap is particularly disadvantageous when sample-induced pupil aberrations occur. For example, in liquid-filled sample containers with a small cross-section, such as the individual wells of a microtiter plate, the surface tension of the liquid creates a distinct liquid meniscus, which leads to a displacement of the illumination pupil upon transmission through the sample. Permanent overlap of the light and phase rings is not possible under these circumstances.

[0004] In the differential interference contrast method according to Nomarski, or DIC for short, the illuminating light is split by a Wollaston prism into two coherent partial beams that are laterally offset from one another in the sample. These two partial beams are superimposed and interfered with on the objective side by another Wollaston prism. This polarization-induced splitting of the two partial beams in the DIC method results in birefringence, such as that caused by the bottom of a standard plastic sample container, leading to a significant deterioration in contrast due to the associated influence on polarization. Since this undesirable birefringence cannot be avoided in the manufacture of conventional plastic sample containers, the Nomarski DIC method cannot be used in plastic Petri dishes or plastic microtiter plates, for example.

[0005] Furthermore, there is a rapidly expanding field of computer-implemented transmitted-light contrast techniques, for example, a technique called differential phase contrast, described in L. Tian & L. Waller, Opt. Exp. 23 (2015), 11394 , a method called transport-of-intensity imaging, described in M. Teague, J. Opt. Soc. Am. 73 (1983), 1434 , and a method called pupil modulation contrast, described in H. Lu et al., Opt. Exp. 24 (2016), 23545 . These methods are based on the fact that by knowing the optical transfer function, see for example Born & Wolf, Principles Of Optics, 7th ed. ISBN 978-0-521-6422-4, 10.6.3 . Imaging with partially coherent quasi-monochromatic illumination,or its linear approximation, which is also known as the weak object transfer function and is described, for example, in N. Streibl, J. Opt. Soc. Am. A 2 (1985), 121, a quantitative reconstruction of phase and absorption information of a sample is possible. This can be done in both two and three dimensions. The disadvantage of these methods is that they require a certain ratio of illumination aperture to detection aperture. For differential phase contrast, the illumination aperture must be larger than or equal to the detection aperture. For pupil modulation contrast, the illumination aperture must be smaller than or equal to the detection aperture.

[0006] However, the aforementioned computer-implemented transmitted-light contrasting methods have several disadvantages. For example, differential phase contrast requires complete illumination of the objective pupil by the transmitted-light illumination system if small to negligible amounts of the phase transfer function are to be avoided at low spatial frequencies. Therefore, this method cannot be universally applied in a compound microscope at a reasonable cost, especially when using high-aperture immersion objectives, since complete illumination of the objective pupil is associated with considerable effort. For transport-of-intensity imaging, images must be acquired at different defoci of the sample. Regardless of whether this defoci is achieved by a classic focusing drive, i.e.If contrast imaging is to be achieved by moving a stage or nosepiece, using a focusable lens, for example a liquid-based lens, wavefront manipulators, such as spatial light modulators (SLMs), or deformable mirrors, multiple images must be acquired sequentially. This reduces the image acquisition rate and may generate image artifacts in dynamic samples. While implementation with multiple differently focused cameras is conceivable, it is complex and may not utilize the full magnification range of the compound microscope. Pupil modulation contrast requires a coherent light source, which is costly and requires additional safety measures to ensure laser safety. Furthermore, coherent illumination reduces the achievable resolution.

[0007] For the state of the art, see also Z. Phillips et al., PLoS ONE 12 (2017), e0171228 ; W. Lee et al., Opt. Exp. 25 (2017), 8398, discloses a method for differential phase contrast in which images are acquired simultaneously. This is achieved by spectral coding of an illumination pupil. However, this method also has the disadvantages mentioned in connection with differential phase contrast.

[0008] Furthermore, with regard to the state of the art, reference is made to the documents DE 10 2013 110497 A1, US 2014 / 354983 A1 and US 2017 / 276613 A1 as well as the documents Hangwen Lu et al, "Quantitative phase imaging and complex field reconstruction by pupil modulation differential phase contrast", OPTICS EXPRESS, US, (20161031), vol. 24, no. 22, page 25345 and Zachary F. Phillips et al, "Single-shot quantitative phase microscopy with colormultiplexed differential phase contrast (cDPC)", PLOS ONE, (20170202), vol. 12, no. 2, page e0171228.

[0009] The object of the present invention is to provide a microscopic transmitted-light contrasting method that can be used across the accessible magnification and aperture range of a compound microscope. Furthermore, the object of the invention is to provide a device with which a corresponding microscopic transmitted-light contrasting method can be carried out.

[0010] This object is achieved by a method having the features of claim 1 and by a device having the features of claim 12. Advantageous further developments are specified in the subclaims.

[0011] In the microscopic transmitted light contrasting method according to the invention, a sample is at least partially illuminated by an asymmetric first illumination pupil and the sample is at least partially imaged by an asymmetric first detection pupil in order to generate a first partial image, wherein the first illumination pupil and the first detection pupil are rotated relative to one another in projection onto a plane lying perpendicular to an optical axis and are arranged to partially overlap one another in such a way that at least a first region of the angular space lies in a bright field and a second region of the angular space, which is different from the first region, lies in a dark field.Furthermore, the sample is at least partially illuminated by an asymmetric second illumination pupil, and the sample is at least partially imaged by an asymmetric second detection pupil to generate a second partial image, wherein the second illumination pupil and the second detection pupil are rotated relative to one another in projection onto the plane perpendicular to the optical axis and are arranged to partially overlap one another such that at least a third region of the angular space lies in a bright field and a fourth region of the angular space, different from the third region, lies in a dark field. An image of the sample is generated from the first partial image and the second partial image by reconstructing a complex transmission function of the sample from the first partial image and the second partial image.

[0012] In this application, angular space refers to the spatial frequency spectrum of the two partial images, which is also referred to as the angular spectrum, particularly in plane wave optics. Thus, angular space represents the space of the Fourier transforms of the two partial images.

[0013] In the present application, the term "asymmetric" refers to the optical axis of an objective assigned to the two detection pupils. This means that the pupils designated as asymmetric are not, as is otherwise common in transmitted-light microscopy, rotationally symmetric with respect to the optical axis of the objective assigned to the two detection pupils. For example, the two illumination pupils and the two detection pupils each have the shape of a circular sector, a circular segment, or an arc of a circle. The respective rotation of the two illumination pupils and the two detection pupils relative to each other is also referred to below as polar rotation. The term "polar" refers to a polar coordinate system whose polar direction is defined by the optical axis of the objective.

[0014] Viewed from the objective lens, the first illumination pupil and the first detection pupil, as well as the second illumination pupil and the second detection pupil, partially overlap. This ensures that the first and second partial images each have a bright-field component, i.e., a light background, and a dark-field component, i.e., a dark background. The illumination pupils thus realize asymmetrical illumination of the sample.

[0015] The bright-field portion of the first and second partial images depict high-contrast, colorful details of the sample—so-called amplitude objects or structures. The dark-field portion, on the other hand, depicts transparent or nearly transparent details of the sample—so-called phase objects or structures—that merely change the phase of the light rays passing through them. Accordingly, the dark-field portion ensures the phase sensitivity of the method. By combining the bright-field and dark-field portions of the first and second partial images, both phase and amplitude information of the sample can be displayed in the image generated from the partial images. By combining phase and amplitude information, a high-contrast image of the sample can be generated.Since the image of the sample is composed of multiple partial images and thus does not require the illumination of an entire objective pupil, the method according to the invention can be used with both high-aperture and low-aperture objectives. In particular, it is irrelevant in the method according to the invention whether the illumination aperture is larger or smaller than the aperture of the objective. This is particularly advantageous in compound microscopes with interchangeable objectives. Furthermore, the method according to the invention can be implemented with only a single sensor element, allowing the full magnification range of the compound microscope to be utilized.

[0016] It goes without saying that the method according to the invention can also generate more than two partial images from which the image of the sample is generated. Each additional partial image is assigned a further asymmetric illumination pupil and a further asymmetric detection pupil, which are arranged relative to one another such that at least one region of the angular space lies in a bright field, and a further region of the angular space, different from this region, lies in a dark field.

[0017] In an advantageous development, the sample is at least partially illuminated with first illumination light through the first illumination pupil, and the sample is at least partially illuminated with second illumination light through the second illumination pupil, wherein the first illumination light and the second illumination light have a different spectral composition. Preferably, the first partial image and the second partial image are recorded simultaneously. This prevents the creation of image artifacts due to a time-shifted recording of the first partial image and the second partial image, particularly when recording dynamic processes in the sample. In this context, one also speaks of spectral coding of the respective illumination pupil. This allows the regions of the angular space through the two illumination pupils to be separated from one another in the two partial images.This separation can be achieved, for example, by a Bayer matrix in front of a CCD or CMOS camera, or by a spectrally splitting prism arrangement in front of a plurality of monochrome CCD or CMOS cameras, or by using dichroic beam splitter plates.

[0018] In a further advantageous development, an adjustment of the sampling, i.e., the sampling rate, noise reduction, or spectral redistribution is performed for the first partial image and / or the second partial image. Before further processing to generate the image of the sample takes place, various image processing operations can be applied to the first partial image and / or the second partial image to improve the image quality of the two partial images and the resulting image of the sample.

[0019] Preferably, the image of the sample is generated by numerically reconstructing a complex transmission function of the sample from the first partial image and the second partial image. The complex transmission function of the sample, also referred to as the complex scattering function, includes phase and amplitude information of the sample. It can be calculated numerically using the phase and amplitude transfer functions of the device used for imaging, using methods known in the art, for example, using a Wiener filter or iterative methods, such as those described in M. Chen et al., Biomed. Opt. Exp. 7 (2016), 3940.

[0020] In an advantageous further development, the steps for generating the image are repeated for different layers of the sample, and an image stack is generated from the images thus generated.

[0021] In a further advantageous development, an illumination profile of the first illumination pupil and / or an illumination profile of the second illumination pupil is recorded. This allows adaptive adjustment of the asymmetric illumination, in particular of the angular spectrum, by comparing a target value of the illumination profile with the recorded actual value of the illumination profile and by adjusting the first illumination pupil and / or the second illumination pupil.

[0022] In an advantageous development, an image of the first illumination pupil and / or the second illumination pupil is generated after transmission through the sample, and phase and amplitude transfer functions are dynamically calculated using this image. The dynamically generated phase and amplitude transfer functions - also called optical transfer functions - can serve in particular as a basis for reconstructing the complex transmission function of the sample. The use of dynamically generated phase and amplitude transfer functions has the advantage over the use of predetermined phase and amplitude transfer functions that the reconstruction is independent of short-term changes in the optical system used for imaging. The generation of a dynamic transfer function can also enable extrapolation orInclude modelling of the illumination spectrum in the dark field, since this cannot be imaged for measuring the illumination spectrum, for example, by a border lens system.

[0023] In a further advantageous development, the first illumination pupil, the second illumination pupil, the first detection pupil and / or the second detection pupil are shaded.

[0024] Preferably, the image generation steps are repeated for different shadings of the first illumination pupil, the second illumination pupil, the first detection pupil, and / or the second detection pupil. Such sequential acquisition reduces crosstalk between color-coded channels and improves the signal-to-noise ratio, which has a decisive influence on the quality of the generated image of the sample.

[0025] Preferably, the first illumination pupil, the second illumination pupil, the first detection pupil and / or the second detection pupil each have the shape of a circular sector or a circular segment.

[0026] It is advantageous if the first illumination pupil and the second illumination pupil are arranged relative to one another in such a way that they can be made to overlap by rotation, and / or if the first detection pupil and the second detection pupil are arranged relative to one another in such a way that they can be made to overlap by rotation. The first partial image and the second partial image thus have a simple geometric relationship to one another, making it particularly easy to generate the image of the sample from the first partial image and the second partial image.

[0027] The invention further relates to a device for carrying out a microscopic transmitted-light contrasting method. The device comprises an illumination unit configured to at least partially illuminate a sample through an asymmetric first illumination pupil and to at least partially illuminate the sample through an asymmetric second illumination pupil, a detection unit configured to at least partially image the sample through an asymmetric first detection pupil to generate a first partial image, and to at least partially image the sample through an asymmetric second detection pupil to generate a second partial image, wherein the first illumination pupil and the first detection pupil are rotated relative to one another and partially overlap one another in projection onto a plane perpendicular to an optical axis,that at least a first region of the angular space lies in a bright field and a second region of the angular space, different from the first region, lies in a dark field, and the first partial image has a first bright field component and a first dark field component, and wherein the second illumination pupil and the second detection pupil, in projection onto the plane perpendicular to the optical axis, are rotated relative to one another and arranged to partially overlap one another such that at least a third region of the angular space lies in a bright field and a fourth region of the angular space, different from the third region, lies in a dark field, and the second partial image has a second bright field component and a second dark field component, and a control unit which is designed to generate an image of the sample from the first partial image and the second partial image,by reconstructing a complex transmission function of the sample from the first partial image and the second partial image.

[0028] Preferably, the illumination unit comprises a first modulator element for generating the first illumination pupil and the second illumination pupil, and the detection unit comprises a second modulator element for generating the first detection pupil and the second detection pupil. The first and second modulator elements physically generate the first and second illumination pupils, or the first and second detection pupils, in the manner of an aperture. The first modulator element and / or the second modulator element can be different apertures, a system of interchangeable apertures, a micromirror array, or spatial light modulators (SLMs). Alternatively, the first illumination pupil and the second illumination pupil can also be generated by LED arrays.

[0029] In an advantageous development, the device comprises a Bertrand lens system for detecting the illumination profile of the first detection pupil and / or the illumination profile of the second detection pupil. The Bertrand lens system allows conoscopic observation of the first detection pupil and / or the second detection pupil. This enables adaptive adjustment of the asymmetric illumination by adjusting the first illumination pupil and / or the second illumination pupil, for example, by controlling the first modulator element.

[0030] Preferably, the illumination unit comprises an incoherent light source. Incoherent light sources are more cost-effective than coherent light sources, i.e., laser light sources. Furthermore, complex measures to ensure laser safety are not required.

[0031] The invention further relates to a microscope, in particular a transmitted-light microscope, having a device according to the type described above. The microscope can be designed either as an upright microscope or as an inverted microscope.

[0032] The invention is explained below with reference to the figures, in which: Figure 1 shows a schematic representation of a transmitted light microscope as a first embodiment; Figure 2 shows a schematic representation of the position of a first illumination pupil and a first detection pupil relative to each other in the transmitted light microscope according to Figure 1 ; Figure 3 an amplitude transfer function of the transmitted light microscope according to Figure 1 ; Figure 4 a phase transfer function of the transmitted light microscope according to Figure 1 ; Figure 5 shows a phase transfer function of an image composed of partial images; Figure 6 shows three partial images of the sample, which were obtained with the aid of the transmitted light microscope after Figure 1were recorded; Figure 7 shows the amplitude structures and phase structures of the sample reconstructed from the three partial images using a Wiener filter; Figure 8 shows a schematic representation of a transmitted-light microscope as a second exemplary embodiment; Figure 9 shows a schematic representation of a transmitted-light microscope with a Bertrand lens system as a third exemplary embodiment; and Figure 10 shows a flow chart showing a specific embodiment of the microscopic transmitted-light contrasting method according to the invention.

[0033] Figure 1 shows a schematic representation of a transmitted-light microscope 10 as a first embodiment. The transmitted-light microscope 10 comprises a device 12 for performing a microscopic transmitted-light contrasting method and an objective 14 directed at a sample 16. The device 12 comprises an illumination unit 18 for illuminating the sample 16, a detection unit 20, and a control unit 22.

[0034] The illumination unit 18 comprises a light source 24 for generating incoherent illumination light 26, a first modulator element 28, which is arranged in a pupil plane of the illumination device 18 and is formed, for example, by a system of replaceable diaphragms, a micromirror arrangement or a spatial light modulator, and a condenser 30. The illumination light 26 generated by the light source 24, after passing through the first modulator element 28, falls on the condenser 30, which directs the illumination light 26 onto the sample 16.

[0035] The sample 16 scatters and / or attenuates the illumination light 26, thereby converting the illumination light 26 into detection light 27, which is used to image the sample 16. The detection light 27 emanating from the sample 16 is directed through the lens 14 into the detection unit 20.

[0036] The detection unit 20 comprises a second modulator element 32, which is arranged in a pupil plane of the objective 14 and is formed, for example, by a system of interchangeable apertures, a micromirror array, or a spatial light modulator. If the objective 14 does not have a pupil plane located outside the objective 14, the pupil plane located inside the objective 14 can be imaged realistically outside the objective 14 using a suitable relay system, and the second modulator element 32 can be positioned in this real interpupil plane. The detection unit 20 further comprises a tube lens 34 and a spatially resolved detector element 36, for example, a CDD or CMOS element.

[0037] The detection light 27 emanating from the sample 16 is first directed by the objective 14 onto the second modulator element 32. After passing through the second modulator element 32, the detection light 27 passes through the tube lens 34 onto the detector element 36. In the case of single-stage imaging with an objective 14 corrected for a finite tube length, the tube lens can also be omitted.

[0038] The control unit 22 controls the first modulator element 28 to generate a first illumination pupil 38, which in Figure 2 is shown in more detail, and a second illumination pupil. As a result, the control unit 22 dynamically controls which areas of the angular space are illuminated by the illumination light 26. The control unit 22 also controls the second modulator element 32 to generate a first detection pupil 40, which in Figure 2shown in more detail, and a second detection pupil. As a result, the control unit 22 dynamically controls which regions of the angular space are imaged by the detection unit 20. Furthermore, the control unit 22 controls the detector element 36 such that a first partial image, which is associated with the first illumination pupil 38 and the first detection pupil 40, and a second partial image, which is associated with the second illumination pupil and the second detection pupil, are generated, and that an image of the sample 16 is generated from the first partial image and the second partial image.

[0039] In the first exemplary embodiment, only two partial images are acquired to generate the image of sample 16. However, more than two partial images can also be acquired. For this purpose, the control unit 22 controls the first modulator element 28 to generate additional illumination pupils and the second modulator element 32 to generate additional detection pupils. Furthermore, the control unit 22 controls the detector element 36 such that additional partial images are generated, each associated with an additional illumination pupil and an additional detection pupil, and that the image of sample 16 is generated from the first partial image, the second partial image, and the additional partial images.

[0040] Figure 2 shows a schematic representation of the position of the first illumination pupil 38 and the first detection pupil 40 relative to each other in the transmitted light microscope according to Figure 1 . The representation in Figure 2shows the pupils 38, 40 seen from the lens 14.

[0041] The first illumination pupil 38 and the first detection pupil 40 are arranged such that they partially overlap. This creates a first illumination region 42, which provides dark-field illumination, and a second illumination region 44, which provides bright-field illumination. The first partial image imaged by the first detection pupil 40 thus has a bright-field component and a dark-field component.

[0042] In the Figure 2In the embodiment shown, the aperture of the first detection pupil 40 is twice as large as the aperture of the first illumination pupil 38. In an alternative embodiment, the aperture of the first detection pupil 40 can also be smaller than the aperture of the first illumination pupil 38. The first illumination pupil 38 and the first detection pupil 40 each have the shape of a circular sector, with the central angle having the value 120°. By rotating by 120°, the second illumination pupil and the second detection pupil can thus be easily generated from the first illumination pupil 38 and the first detection pupil 40. In the embodiment shown in Figure 2 In the embodiment shown, the first illumination pupil 38 and the first detection pupil 40 are polar rotated relative to each other by an angle of 60°.

[0043] Should be based on Figure 1 and 2If the entire angular space is to be imaged using the transmitted light microscope 10 shown, a third partial image must be generated which is assigned to a third illumination pupil and a third detection pupil, which are each generated by a rotation of 240° from the first illumination pupil 38 and the first detection pupil 40, respectively.

[0044] Figure 3 shows an amplitude transfer function 46 of the transmitted light microscope 10 according to Figure 1 for a partial image. The amplitude transfer function 46 is a function in angular space and specifies how the magnitudes of spatial frequencies are transmitted through the transmitted-light microscope 10.

[0045] Figure 4 shows a phase transfer function 48 of the transmitted light microscope 10 according to Figure 1for a partial image. The phase transfer function 48, like the amplitude transfer function 46, is a function in angular space and specifies how the phases of spatial frequencies are transmitted through the transmitted-light microscope 10. It has a zero crossing 50 typical for asymmetric illumination.

[0046] Figure 5 shows a phase transfer function 52 of the image of the sample 16 generated from the partial images. The Figure 5 It can be seen that by combining several partial images of the sample 16, the range of spatial frequencies imaged by the transmitted light microscope 10 is increased and the zero crossings 50 of the phase transfer functions of the partial images are removed down to the origin of the angular space, which leads to an improvement in the contrast of the image of the sample 16.

[0047] Figure 6 shows three partial images 54a, 54b, 54c generated by an optical simulation, which are examined with the aid of the transmitted light microscope 10 according to Figure 1 were recorded.

[0048] A first partial image 54a of the three partial images 54a, 54b, 54c was generated by the Figure 2 illustrated first illumination pupil 38 is illuminated and through the also shown Figure 2illustrated first detection pupil 40. A second partial image 54b of the three partial images 54a, 54b, 54c was illuminated by the second illumination pupil, which was generated by rotating the first illumination pupil 38 by 120° about the optical axis of the objective 14, and imaged by the second detection pupil, which was generated by rotating the first detection pupil 40 by 120° about the optical axis of the objective 14. A third partial image 54c of the three partial images 54a, 54b, 54c was illuminated by the third illumination pupil, which was generated by rotating the first illumination pupil 38 by 240° around the optical axis of the objective 14, and imaged by the third detection pupil, which was generated by rotating the first detection pupil 40 by 240° around the optical axis of the objective 14.

[0049] Each of the three partial images 54a, 54b, 54c shows the sample 16, which has both an amplitude structure 56 and a transparent phase structure 58.

[0050] Figure 7 shows the amplitude structures 56 and phase structures 58 of sample 16 reconstructed from the three partial images 54a, 54b, 54c using a Wiener filter.

[0051] Figure 8 shows a schematic representation of a transmitted light microscope 60 as a second embodiment. Figure 8 The transmitted light microscope 60 shown differs from the one in Figure 1 shown transmitted light microscope 10 essentially in that the control unit 22 does not dynamically control the first modulator element 66 and the second modulator element 70.

[0052] The transmitted light microscope 60 after Figure 8comprises the device 62 for carrying out the transmitted light contrasting method according to the invention. The device 62 comprises the illumination unit 64 for illuminating the sample 16 and the detection unit 70. Identical or equivalent elements are shown in the Figure 1 and 8 designated by the same reference symbol.

[0053] The illumination unit 64 comprises the first modulator element 66, which is arranged in a pupil plane of the illumination device 18 and is designed to spectrally encode the illumination light 26 emanating from the light source 24 according to the first illumination pupil 38 and the second illumination pupil. This can be done, for example, with the aid of several spectral filters, each of which is designed in the shape of the first illumination pupil 38 and the second illumination pupil, respectively.

[0054] The detection unit 68 comprises the second modulator element 70, which is arranged in the pupil plane of the objective 14 and is designed to spectrally encode the detection light 27 emanating from the sample 16 according to the first detection pupil 40 and the second detection pupil, as well as the spatially resolved detector element 72. The spatially resolved detector element 72 is designed to separate the detection light 27 into two partial images. This separation is achieved using a Bayer matrix.

[0055] The control unit 22 controls the detector element 72 such that the first partial image, which is assigned to the first illumination pupil 38 and the first detection pupil 40, and the second partial image, which is assigned to the second illumination pupil and the second detection pupil, are generated simultaneously and that the image of the sample 16 is generated from the first partial image and the second partial image.

[0056] Figure 9shows a schematic representation of a transmitted light microscope 74 as a third embodiment. Figure 9 The transmitted light microscope 74 shown differs from the one in Figure 1 shown transmitted-light microscope 10 essentially in that the device 78 for carrying out the transmitted-light contrasting method according to the invention additionally comprises a Bertrand lens system 76. Identical or equivalent elements are shown in the Figure 1 and 9 designated by the same reference symbol.

[0057] The Bertrand lens system 76 comprises a beam splitter 80 arranged in a focal plane of the objective 14, with the aid of which a portion of the illumination light 27 is directed onto another spatially resolved detector element 82. The Bertrand lens system 76 enables the detection of the illumination profile of the detection pupils. This, in turn, allows the control unit 22 to adaptively adjust the angular spectrum of the illumination by the illumination unit 18 by controlling the modulator element 28.

[0058] Identical or equivalent features are listed in the Figure 1 and 9 designated by the same reference symbol.

[0059] Figure 10 shows a flow chart showing a specific embodiment of the microscopic transmitted light contrasting method according to the invention.

[0060] In a first step S1, a total of N partial images are recorded. The partial images are each assigned to exactly one illumination pupil and one detection pupil. The illumination pupil and the detection pupil are each arranged in such a way that at least one region of the angular space lies in a bright field and another region of the angular space, different from this region, lies in a dark field. Each of the N partial images thus has a bright field component and a dark field component. The recording of the N partial images can be carried out sequentially, i.e. one after the other in time, or simultaneously, i.e. at the same time. If the recording is carried out simultaneously, a spectral coding of the illumination pupil must be carried out in each case, as described above in connection with Figure 8 is described in more detail.

[0061] In a second step S2, the N partial images are preprocessed, i.e., for each partial image, for example, the sampling is adjusted, noise reduction is performed, and / or spectral redistribution is performed. This serves to improve the image quality of the image of sample 16 reconstructed from the N partial images.

[0062] In a third step S3, the complex transmission function, i.e. the complex scattering potential, of the sample 16 is reconstructed as an image of the sample 16 from the N partial images and the associated transfer functions, i.e. the optical transmission functions. This can be done, for example, using a Wiener filter or iterative methods, as described, for example, in M. Chen et al., Biomed. Opt. Exp. 7 (2016), 3940. An example of such a reconstruction is shown in the Figure 6 and 7shown. The complex transmission function of sample 16 can be represented in particular as separate amplitude and phase distributions. List of reference symbols

[0063] 10Transmitted-light microscope 12Device 14Objective lens 16Specimen 18Illumination unit 20Detection unit 22Control unit 24Light source 26Illumination light 27Detection light 28Modulator element 30Condenser 32Modulator element 34Tube lens 36Detector element 38Illumination pupil 40Detection pupil 42, 44Area 46, 48Transfer function 50Zero crossing 52Transfer function 54a, 54b, 54cPartial image 56Amplitude object 58Phase object 60Transmitted-light microscope 62Device 64Illumination unit 66Modulator element 68Detection unit 70Modulator element 72Detector element 74Transmitted-light microscope 76Bertrand lens system 78Device 80Beam splitter 82Detector element

Claims

1. Microscopic transmitted light contrasting method, in which a sample (16) is at least partially illuminated by an asymmetric first illumination pupil (38), the sample (16) is at least partially imaged by an asymmetric first detection pupil (40), to generate a first partial image (54a), wherein the first illumination pupil (38) and the first detection pupil (40) are rotated relative to each other in projection onto a plane, perpendicular to an optical axis, and are arranged to partially overlap each other, in such a way that at least a first region of the angular space lies in a bright field and a second region of the angular space, different from the first region, lies in a dark field and the first partial image (54a) has a first bright field portion and a first dark field portion, the sample (16) is at least partially illuminated by an asymmetric second illumination pupil, the sample (16) is at least partially imaged by an asymmetric second detection pupil to produce a second partial image (54b), wherein the second illumination pupil and the second detection pupil are rotated relative to each other in projection onto the plane perpendicular to the optical axis and are arranged to partially overlap each other, such that at least a third region of the angular space lies in a bright field and a fourth region of the angular space, different from the third region, lies in a dark field and the second partial image has a second bright field portion and a second dark field portion, and an image of the sample is generated from the first partial image (54a) and the second partial image (54b) by reconstructing a complex transmission function of the sample (16) from the first partial image (54a) and the second partial image (54b).

2. The method according to claim 1, characterised in that the sample (16) is at least partially illuminated with first illumination light through the first illumination pupil (38), the sample (16) is at least partially illuminated with second illumination light through the second illumination pupil, and the first illumination light and the second illumination light have a different spectral composition.

3. The method according to claim 2, characterised in that the first partial image (54a) and the second partial image (54b) are generated simultaneously.

4. The method according to any one of claims 1 to 3, characterised in that an adaptation of the sampling, a noise reduction or a spectral redistribution is carried out for the first partial image (54a) and / or the second partial image (54b).

5. The method according to any one of claims 1 to 4, characterised in that the image of the sample (16) is generated by numerically reconstructing the complex transmission function of the sample (16) from the first partial image (54a) and the second partial image (54b).

6. The method according to any one of claims 1 to 5, characterised in that the steps for generating the image are repeated for different layers of the sample (16) and an image stack is generated from the images thus generated.

7. The method according to any one of claims 1 to 6, characterised in that an illumination profile of the first illumination pupil (38) and / or an illumination profile of the second illumination pupil is detected.

8. The method according to any one of claims 1 to 7, characterised in that an image of the first illumination pupil (38) and / or the second illumination pupil is generated after transmission through the sample (16) and phase and amplitude transfer functions (46, 48) are calculated using this image.

9. The method according to any one of claims 1 to 8, characterised in that the first illumination pupil (38), the second illumination pupil, the first detection pupil (40) and / or the second detection pupil are shaded.

10. The method according to claim 9, characterised in that the steps for generating the image are repeated for different shadings of the first illumination pupil (38), the second illumination pupil, the first detection pupil (40) and / or the second detection pupil.

11. The method according to any one of claims 1 to 10, characterised in that the first illumination pupil (38), the second illumination pupil, the first detection pupil (40) and / or the second detection pupil each have the shape of a circular sector or a circular segment.

12. The method according to any one of claims 1 to 11, characterised in that the first illumination pupil (38) and the second illumination pupil are arranged relative to each other in such a way that they can be brought into overlap by rotation, and / or the first detection pupil (38) and the second detection pupil are arranged relative to each other in such a way that they can be brought into overlap by rotation.

13. Apparatus for carrying out a microscopic transmitted light contrasting method, comprising: an illumination unit (18, 64) configured to illuminate a sample (16) at least partially through an asymmetric first illumination pupil (38) and to illuminate the sample (16) at least partially through an asymmetric second illumination pupil, a detection unit (20, 68), configured to image the sample (16) at least partially through an asymmetric first detection pupil (40) to generate a first partial image (54a) and to image the sample (16) at least partially through an asymmetric second detection pupil to generate a second partial image (54b), wherein the first illumination pupil (38) and the first detection pupil (40) are rotated relative to each other in projection onto a plane, perpendicular to an optical axis, and are arranged to partially overlap each other, in such a way that at least a first region of the angular space lies in a bright field and a second region of the angular space, different from the first region, lies in a dark field and the first partial image (54a) has a first bright field portion and a first dark field portion, and wherein the second illumination pupil and the second detection pupil are rotated relative to each other in projection onto the plane perpendicular to the optical axis and are arranged to partially overlap each other, such that at least a third region of the angular space lies in a bright field and a fourth region of the angular space, different from the third region, lies in a dark field and the second partial image (54b) has a second bright field portion and a second dark field portion, and a control unit (22) which is configured to generate an image of the sample (16) from the first partial image (54a) and the second partial image (54b) whereby the control unit (22) reconstructs a complex transmission function of the sample (16) from the first partial image (54a) and the second partial image (54b).

14. The apparatus according to claim 13, characterised in that the illumination unit (18, 64) comprises a first modulator element (28, 66) for generating the first illumination pupil (38) and the second illumination pupil, and the detection unit (20, 68) comprises a second modulator element (32, 70) for generating the first detection pupil and the second detection pupil.

15. The apparatus according to claim 13 or 14, characterised by a Bertrand lens system (76) for detecting an illumination profile of the first detection pupil (38) and / or an illumination profile of the second detection pupil.