WO2018197230A1

WO2018197230A1 - Phase contrast imaging having a transmission function

Info

Publication number: WO2018197230A1
Application number: PCT/EP2018/059452
Authority: WO
Inventors: Lars STOPPE; Thomas Milde
Original assignee: Carl Zeiss Microscopy Gmbh
Priority date: 2017-04-26
Filing date: 2018-04-12
Publication date: 2018-11-01
Also published as: CN110520780A; CN110520780B; DE102017108873A1

Abstract

The invention relates to an optical system (100), which comprises: a sample holder (113) which is configured in order to fix a sample object; and an illumination module (111) which is configured in order to illuminate the sample object with at least one structured illumination geometry (300); and an optical imaging system (112) which is configured in order to produce, on a detector (114), an image of the sample object illuminated by the at least one structured illumination geometry (300); and the detector (114) which is configured in order to capture at least one picture of the sample object based on the image; and a controller (115) which is configured in order to determine a resulting picture based on a transmission function (400) and the at least one picture, the resulting picture having a phase contrast. In this case the transmission function (400) corresponds to a scaled reference transmission function (400) based on a size of an aperture of the optical imaging system (112).

Description

Phase-contrast imaging with transfer function

TECHNICAL FIELD Various examples of the invention generally relate to an optical system having a lighting module configured to illuminate a sample object having a structured illumination geometry. Various examples of the invention relate in particular to techniques based on a

Transfer function and at least one image of the sample object to determine a result image having a phase contrast.

BACKGROUND

In optical imaging of sample objects, it may often be desirable to create a so-called phase contrast image of the sample object. In one

Phase contrast image is at least a part of the image contrast by a

Phase shift of the light caused by the imaged sample object. In particular, such sample objects can be imaged with comparatively high contrast, which cause no or only a slight weakening of the amplitude, but a significant phase shift; Such sample objects are often referred to as phase objects. Typically, biological samples as a sample object in a microscope can cause a comparatively larger phase change than an amplitude change of the electromagnetic field. There are several techniques for phase-contrast imaging, such as

Dark field illumination, the oblique illumination, the differential interference contrast (DIC) or the Zernike phase contrast.

Such aforementioned techniques have several disadvantages or limitations. Often it may be necessary to provide additional optical elements between the sample and the detector in the region of the so-called imaging optics, in order to obtain the

Enable phase-contrast imaging. This can be constructive Restrictions result. Furthermore, application restrictions may exist: For example, fluorescence imaging may be hampered by providing the additional optical elements. There are also known techniques in which a phase contrast can be achieved by means of structured illumination. A first example of techniques which can achieve an image with phase contrast by means of structured illumination is disclosed in DE 10 2014 1 12 242 A1. However, such techniques have certain

Restrictions on. For example, by means of such techniques in which different intensity images associated with different illumination directions are combined, it may be possible for a corresponding result image to represent the phase contrast gradient-like. This means that opposite edges of a phase object can have a different sign of contrast. Thus, it may not or only partially be possible by means of such techniques, for example to produce height profiles of the sample object, which a

Contrast having large (small) values for high (deep) points of the sample object.

Another technique in which a result image with phase contrast can be achieved by means of structured illumination is the so-called quantitative difference

Phase contrast technique (English, quantitative differential phase contrast, QDPC). See, for example, L. Tian and L. Waller: "Quantitative differential phase contrast imaging in an LED array microscope", Optics Express 23 (2015), 1 1394 (hereinafter Tian, Waller), however, such techniques have the disadvantage that, depending on For example, certain requirements with respect to the size of an aperture of the lighting module relative to a size of an aperture may be optic-to-detection

Restrict tasks to use. In addition, it has been observed that undesirable amplification of imaged frequencies may occur in the QDPC, eg, if there are some tolerances with respect to the aperture used. BRIEF DESCRIPTION OF THE INVENTION

Therefore, there is a need for improved techniques for determining result images with phase contrast. In particular, there is a need for such techniques that overcome or mitigate at least some of the above limitations and disadvantages.

This object is solved by the features of the independent claims. The features of the dependent claims define embodiments.

In one example, an optical system includes a sample holder. The sample holder is arranged to fix a sample object. The optical system also includes a lighting module. The illumination module is set up to illuminate the sample object with at least one structured illumination geometry. The optical system also includes imaging optics configured to generate an image of the sample object illuminated with the at least one structured illumination geometry on a detector. The optical system also includes the detector. The detector is configured to generate at least one image of the image based on the image

To capture a sample object. The optical system also includes a controller. The controller is configured to determine a result image based on a transfer function and the at least one image. The result image has a phase contrast. In this case, the transfer function corresponds to a reference transfer function scaled based on a size of an aperture of the imaging optics.

In one example, an optical system includes a sample holder. The sample holder is arranged to fix a sample object. The optical system also includes a lighting module. The illumination module is set up to illuminate the sample object with at least one structured illumination geometry. The optical system also includes imaging optics configured to generate an image of the sample object illuminated with the at least one structured illumination geometry on a detector. The optical system also includes the detector. Of the Detector is arranged to generate at least one image of the image based on the image

To capture a sample object. The optical system also includes a controller. The controller is configured to determine a result image based on a transfer function and the at least one image. The result image has a phase contrast. A size of the aperture of the illumination module is smaller than a size of the aperture of the imaging optics.

In one example, a method includes illuminating a sample object having at least one structured illumination geometry. The method also includes generating an image of the one having the at least one structured one

Illumination geometry illuminated specimen object. Based on the image, the method further comprises capturing at least one image of the sample object. Based on a transfer function and the at least one image, a result image is determined which has a phase contrast. The

Transfer function corresponds to a reference transfer function that is scaled based on a size of an aperture of the imaging optics.

A computer program product includes program code that can be executed by at least one processor. Running the program code causes the at least one processor to perform a method. The method includes illuminating a sample object with at least one structured one

Illumination geometry. The method also includes generating an image of the illuminated one with the at least one structured illumination geometry

Sample object. Based on the image, the method further comprises capturing at least one image of the sample object. Based on a transfer function and the at least one image, a result image is determined which contains a

Has phase contrast. The transfer function corresponds to a reference transfer function that is scaled based on a size of an aperture of the imaging optics.

A computer program includes program code that can be executed by at least one processor. Executing the program code causes the at least one processor performs a method. The method includes the

Illuminate a sample object with at least one structured one

By means of such techniques, it may be possible to have result images with one

Phase contrast to determine particularly flexible. Namely, such techniques are based on the knowledge that, by suitably selecting the transfer function, it may be possible to determine the result image with the phase contrast, even if, for example, a particularly large aperture of the imaging optics is used.

Such techniques are based in particular on the recognition that, on the one hand, the result image does not necessarily encode a quantitative description of the phase of the sample object by means of the phase contrast, but with a suitable choice of the transfer function, a qualitative description of the phase of the

Sample object e.g. as height profile provides.

The features and features set out above, which are described below, can be used not only in the corresponding combinations explicitly set out, but also in other combinations or isolated, without departing from the scope of the present invention. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates an optical system according to various examples, the optical system having a lighting module configured to illuminate a sample object having a structured illumination geometry.

FIG. 2 schematically illustrates the lighting module having a plurality of

Lighting elements in greater detail. FIG. 3 schematically illustrates an exemplary illumination geometry that can be used to illuminate the sample object by means of the illumination module.

FIG. 4 schematically illustrates an exemplary illumination geometry that can be used to illuminate the sample object by means of the illumination module.

FIG. 5 schematically illustrates an exemplary illumination geometry that can be used to illuminate the sample object by means of the illumination module.

FIG. 6 schematically illustrates a transfer function that may be used in determining a result image according to various examples.

FIG. 7 schematically illustrates a transfer function that may be used in determining a result image, according to various examples, wherein the transfer function of FIG. 7 with respect to the transfer function according to FIG. 8 is scaled.

FIG. 8 schematically illustrates a transfer function that may be used in determining a result image according to various examples. FIG. 9 schematically illustrates transfer functions that may be used to determine a result image according to various examples. FIG. 10 is a flowchart of an example method.

DETAILED DESCRIPTION OF EMBODIMENTS

The above-described characteristics, features, and advantages of this invention, as well as the manner in which they will be achieved, will become clearer and more clearly understood in connection with the following description of the embodiments, which will be described in detail in conjunction with the drawings.

In the figures, like reference characters designate the same or similar elements. The figures are schematic representations of various embodiments of the invention. Elements shown in the figures are not necessarily drawn to scale. Rather, the different ones are in the figures

represented elements such that their function and general purpose will be understood by those skilled. Connections and couplings between functional units and elements illustrated in the figures may also be implemented as an indirect connection or coupling. A connection or coupling may be implemented by wire or wireless. Functional units can be implemented as hardware, software or a combination of hardware and software.

Hereinafter, techniques will be described to provide a result image

to determine tailor-made contrast. For example, the result image can map a phase object with a phase contrast. The result image can be in

Generally provide a height profile of the sample object.

The techniques described herein make it possible to determine the result image by digital post-processing of one or more images of a sample object. For example, it would be possible for the one or more images of the

Sample object are intensity images which themselves have no phase contrast. The one or more images of the sample object may be associated with different illumination geometries. This means that the one or more images can each be detected by a detector with simultaneous illumination of the sample object by means of a corresponding illumination geometry.

The different lighting geometries can, for example, with

be associated with different lighting directions. The different ones

Illumination geometries or associated different images can be separated from one another by time multiplexing or frequency multiplexing. It would also be possible to separate by means of different polarizations. The

Illumination geometries may have a directionality, for example, the illumination geometries may have a gradient of illuminance along one or more spatial directions. For example, For example, the illuminance could vary in steps along a spatial direction, such as between zero and a finite value, or between two different finite values.

For example, the sample object may comprise a phase object, such as a cell or cell culture, etc. The sample object may be a-priori unknown, i. Different sample objects can be fixed by the sample holder. The sample object could also be non-translucent for the light used. Depending on the type of sample object, it may be desirable to operate the illumination module and the detector in incident light geometry or transmitted light geometry.

In various examples, a transfer function for the digital

Post processing of one or more images used to obtain the result image. For example, the transfer function can be a

Object transfer function and / or an optical transmission function of the optical system denote. The transfer function may be suitable for predicting the at least one image for a specific illumination and a specific sample object. For example, the transfer function may have a real-valued component and / or an imaginary component. In this case, the real-valued component of the transfer function can contribute to a decrease in the intensity of the light Pass through the sample object correspond. An amplitude object typically has significant attenuation of the light. Accordingly, the imaginary portion of the transfer function may be a shift in the phase of the sample object

denote continuous light. A phase object typically has a significant shift in the phase of the light. In the following, techniques are described in particular to determine the imaginary part of the transfer function. For the sake of simplicity, reference will not be made below to the fact that the techniques relate to the imaginary part of the transfer function. In some examples, a purely imaginary transfer function without real valued part may be used.

Different techniques for determining the transfer function can be used. In one example, the transfer function could be determined based on a technique according to Abbe. By means of a technique according to Abbe, a reference transfer function could be determined. In this case, the sample object can be separated into different spatial frequency components. Then an overlay of infinitely many harmonic gratings can model the sample object. The light source can also be decomposed into the sum of different point light sources. Another example relates to the determination of the optics transfer function which describes the image of the sample object for a particular illumination geometry based on a Hopkins technique, see H.H. Hopkins "On the Diffraction Theory of Optical Images," Proceedings of the Royal Society A: Mathematical, Physical Engineering Sciences 217 (1953) 408-432, from which the transmission cross-coefficient matrix (TCC) can be determined sometimes referred to as a partially coherent object transfer function, the TCC may serve as a reference transfer function

Transfer function of the partially coherent image and contains the properties of the optical system and the illumination geometry. The frequencies that transmit the optics are limited to the area in which the TCC assumes values other than 0. A system with a high coherence factor or coherence parameter consequently has a larger area with TCC Φ 0 and is capable of higher

Map spatial frequencies. The TCC typically contains all the information of the optical system and the TCC often takes into account even complex valued pupils such. B. in Zernike phase contrast or triggered by aberrations. The TCC may allow separation of the optics transfer function from the object transfer function. In some examples, it is also possible that the

Transfer function is specified and no determination must be made as TCC or Abbe.

Depending on the transfer function used, different techniques for determining the result image may be used. An exemplary technique is described in Tian, Waller with reference to Eq. 13 described. There, it is shown how, based on a Tichonov regularization, a result image by means of inverse Fourier transformation and based on the transfer function H * and further based on the

Spatial frequency space representation of a combination I _DPC of two images of the

Sample object can be determined with different illumination geometries:

In this case, I _{DPC describes} the spectral decomposition of a combination of two images / approximately IB, which were recorded at different illumination geometries that illuminate mutually complementary semicircles:

These are examples. For example, in general, the illumination geometry need not be strictly semi-circular. For example, four LEDs could be used, which are arranged on a semicircle. For example, could be defined

Lighting directions are used, so some individual LEDs. Furthermore, in Eq. 2 also normalize to 1 instead of I _T + I _B , or to some other value. Instead of a clearing of / r and IB could be in others

Examples are also the raw data itself used, eg I _DPC = I _T or I _DPC = I _B. By forming a corresponding quotient in Eq. 2 may fail disturbing influences such as other material properties, color, etc. are reduced. By forming the difference, in particular, an absorption component due to a real-valued component of the transfer function can be reduced. I _DPC is proportional to the local increase in phase shift due to the sample object. The

Phase shift can be caused by a change in the thickness of the sample object or the topography of the sample object and / or by a change in the optical properties.

For example, two images I _DPC and I _DPC, 2 can be determined, once with a pair of semi-circular illumination geometries that are arranged top-bottom in a lateral plane perpendicular to the beam path (I _D _pc, i) ^an d once with a pair of semicircular illumination geometries arranged left-right in the lateral plane (l _DPC , 2) - Then both I _DPC and I _{DPC> 2} can be taken into account in determining the result image, see Summation Index in Eq. 1 .

Such techniques are based on certain assumptions and simplifications, for example in the case of the above-mentioned. Formulation of a weak object approximation and the TCC. In other examples, however, other approximations and formalisms may be used. For example, another inversion could be used instead of Tichonov regularization, such as direct integration or otherwise-trained Fourier filtering. Even in such modifications, the basic characteristics of the

Transfer function as described in the various examples herein.

FIG. 1 illustrates an example optical system 100. For example, the optical system 100 according to the example of FIG. 1 implement a light microscope, for example in transmitted-light geometry. Such a microscope could for

Phase contrast imaging can be used. In other examples, the optical system 100 according to the example of FIG. 1 also implement a light microscope, in Auflichtgeometrie. For example, a corresponding Light microscope used in Auflichtgeometrie for material testing. For this a height profile of the sample object can be created.

By means of the optical system 100, it may be possible to enlarge small structures of a sample object fixed by a sample holder 13. For example, the optical system 100 could implement a wide field microscope in which a sample is fully illuminated. In some examples, the imaging optics 12 may generate an image of the sample object on a detector 14. The detector 14 may then be configured to capture one or more images of the sample object. A viewing through an eyepiece is conceivable.

In some examples, large aperture imaging optics 12 may be used. For example, imaging optics 112 could have a numerical aperture of not less than 0.2, optionally not less than 0.3, more optionally not less than 0.5. For example, the imaging optics 1 12 could have an immersion objective.

The optical system 100 also includes a lighting module 1 1 1. Das

Illumination module 1 1 1 is set up to the sample object, which on the

Sample holder 1 13 is fixed to light. For example, this lighting could be implemented by means of Köhler illumination. There will be a

Condenser lens and a condenser aperture used. This leads to a particularly homogeneous intensity distribution of the light used for the illumination in the plane of the sample object. For example, a partially incoherent

Lighting be implemented. The lighting module 1 1 1 could also be set up to illuminate the sample object in dark field geometry.

In the example of FIG. 1, the lighting module 1 1 1 is set up to allow structured lighting. This means that by means of

Illumination module 1 1 1 different illumination geometries of the

Illumination of the sample object used light can be implemented. The different illumination geometries can correspond to illumination of the sample object from different illumination directions.

In the various examples described herein, different hardware implementations are possible to accommodate the different ones

To provide illumination geometries. For example, the lighting module 1 1 1 could include a plurality of adjustable lighting elements configured to locally modify or emit light. A controller 1 15 can the

Lighting module 1 1 1 or the lighting elements to implement a specific lighting geometry to control.

For example, the controller 1 15 could be implemented as a microprocessor or microcontroller. Alternatively or additionally, the controller 1 15 could include, for example, an FPGA or ASIC. The controller 1 15 may alternatively or additionally also the sample holder 1 13, the imaging optics 1 12, and / or the detector 1 14 control.

FIG. 2 illustrates aspects relating to the lighting module 1 1 1. In FIG. 2 it is shown that the lighting module 1 1 1 a variety of adjustable

Illumination elements 121 in a matrix structure. The matrix structure is oriented in a plane perpendicular to the beam path of the light (lateral plane;

Spatial coordinates x, y).

Instead of a matrix structure, it would also be possible in other examples to use other geometric arrangements of the adjustable elements, for example ring-shaped, semicircular, etc.

In one example, the adjustable illumination elements 121 could be implemented as light sources, such as light emitting diodes. For example, it would then be possible for different light-emitting diodes with different light intensity to emit light for illuminating the sample object. As a result, a lighting geometry can be implemented. In another implementation, the Illumination module 1 1 1 as a spatial light modulator (English, spatial light modulator, SLM) to be implemented. The SLM can be spatially resolved into an intervention

Condenser pills, which may have a direct impact on imaging - for example, formalized by the TCC.

FIG. FIG. 3 illustrates aspects related to an exemplary illumination geometry 300. In FIG. 3 is the provided luminous intensity 301 for the various adjustable ones

Elements 121 of the illumination module 1 1 1 along the axis Χ-Χ ^' of FIG. 2 shown. The illumination geometry 300 has a dependence on the position along the axis XX 'and is therefore structured.

FIG. 4 illustrates aspects related to an example illumination geometry 300. FIG. 4 illustrates the illumination geometry 300 abstractly from the illumination module 1 1 1 used. In the example of FIG. 4, an illumination geometry 300 is used in which one side is illuminated (black color in FIG. 4) and the other side is not illuminated (white color in FIG. 4). In FIG. 5, another exemplary illumination geometry is shown (with corresponding color coding as already described with respect to FIG. 4). FIG. 6 illustrates aspects relating to an example transfer function 400

(In Fig. 6, black is an integer of +1 and white denotes an amount of -1, and the coordinates u _x and u _y are defined in the spatial frequency space, where they correspond to the spatial coordinates x and y). The transfer function 400 may be used to generate, based on an image, for example, the

Illumination geometry 300 according to the example of FIG. 4 was recorded

Determine the result image. The result image may have a phase contrast. The result image may include a height profile of the sample object.

In the example of FIG. 6, the transmission function 400 has an axis of symmetry 405 which corresponds to an axis of symmetry 305 of the illumination geometry 300. As a result, it may be possible for the transfer function 400 to match the Illumination geometry 300 is selected. As a result, the result image can have a particularly strong contrast.

In FIG. 6, the diameter of the detector aperture of the imaging optics 1 12 is also shown. Because a partially incoherent illumination is used, the

Transfer function up to twice the size of the detector aperture

Imaging optics 1 12 not equal to zero.

FIG. 7 also illustrates aspects relating to a transfer function 400. The example of FIG. 7 basically corresponds to the example of FIG. 6. However, in the example of FIG. 6, the size of the detector aperture is greater than in the example of FIG. 6 (see horizontal dashed lines, NA indicates the size of the detector aperture).

In this case, however, the transfer function 400 is scaled correspondingly to the in FIG. 7 compared to FIG. 6 enlarged detector aperture. For example, the

Transfer function 400 according to the example of FIG. 6 serve as a reference transfer function. Then, for example, the controller 1 15 could be configured to perform the transfer function 400 according to the example of FIG. 7 based on a scaling of this reference transfer function on the enlarged aperture of the imaging optics 1 12 to determine.

Based on such techniques, it is possible that result image with the

Phase contrast to be determined for scenarios in which the size of the aperture of the illumination module 1 1 1 is smaller than the size of the aperture of the imaging optics 1 12. In particular, it may be possible in such examples that a particularly large aperture for the imaging optics 1 12 used becomes what happens at certain

Use cases for imaging by means of the optical system 100 may be desirable. For example, it would be possible for the size of the aperture of the

Illumination module 1 1 1 is less than 50% of the size of the aperture of the imaging optics 1 12, optionally less than 20%, further optionally less than 5%. This makes it possible to measure particularly sensitive. From the examples of FIGS. 6 and 7 it can be seen that it may be possible to transfer function 400 irrespective of the size of the aperture of the

Illumination module 1 1 1 to determine. In other words, this may mean that, for example, an extension or certain features - such as e.g. Extreme values, zeros, inflection points, etc. - the transfer function 400 does not depend on the size of the aperture of the lighting module 1 1 1. For example, the

Transfer functions 400 in the examples of FIGS. 6 and 7 have no features, such as local extreme values or zeros, which depend on the size of the aperture of the illumination module 1 1 1 in the spatial frequency space - i. in the space conjugated to the space - would be positioned. Between the place space and the

Spatial frequency space can be transformed by Fourier analysis and inverse Fourier analysis. Spatial frequencies thereby denote the sweep of a spatial

Period length. Rather, the range within which the transfer function 400 assumes non-zero values is determined by the size of the aperture of the imaging optics 12.

Such techniques are based on the recognition that it is also possible for such transfer functions 400 scaled in dependence on the size of the aperture of the imaging optics to determine the resulting image with a meaningful contrast-for example a phase contrast or with a height profile of the sample object. Incidentally, in some examples, the contrast in the result image may not include a quantitative description of the phase of the sample object, but a qualitative description of the phase of the sample object. In particular, the qualitative description of the phase of the sample object can be consistently provided in the area of the entire image. In particular, in comparison to reference techniques such as e.g. described in DE 10 2014 1 12 242 A1, in which different gradients of the phase of the sample object - for example, on opposite edges of the sample object - are mapped with different signs of contrast in the result image, this may have an advantage.

FIG. Figure 8 illustrates aspects relating to a transfer function 400 (in Figure 8, black encodes an amount of +1 and knows an amount of -1; the coordinates u _x and ty are defined in the spatial frequency space and correspond there the

Spatial coordinates x and y). The transfer function 400 may be used to generate, based on an image, for example, the

Illumination geometry 300 according to the example of FIG. 5 was recorded

To determine the result image with a phase contrast. In FIG. 8 is also the

Diameter of the detector aperture of the imaging optics 1 12 shown.

From the FIGs. 6-8, it can be seen that the transfer function 400 can be determined as a function of the structured illumination geometry 300. In particular, it is possible for the geometry of the transfer function 400 in the spatial frequency space to simulate the illumination geometry 300 in the spatial domain. By such techniques, a particularly strong contrast can be achieved in the resulting image, i. a high signal-to-noise ratio, for example for the phase contrast or the height profile. FIG. 9 illustrates aspects related to various transfer functions 400

(Different transfer functions are shown in Fig. 9 by the solid line, the dashed line, the dotted line, and the dashed-dotted line). The in FIG. 9 can be used for different illumination geometries, for example (in FIG

Illumination geometries not shown).

In FIG. 9 is the transfer function 400 along an axis u _x of

Spatial frequency space shown. In some examples, the transfer function could only vary along a coordinate of the spatial frequency space; in other examples, however, there could be variation along two orthogonal axes u _x and u _y . In the example of FIG. 9, for example, a transfer function 400 is formed as a monotonically increasing linear function (solid line). In addition, in the example of FIG. 9 shows another transfer function 400 as a monotone increasing Sigmoid function formed (dashed line). In the example of FIG. 9, another transfer function 400 is formed as a folded, monotonically decreasing, linear function (dotted line). In the example of FIG. 9 is another

Transfer function 400 formed as a step function (dotted line).

Such embodiments of transfer functions 400 are purely exemplary, and in other examples, differently configured transfer functions may be used or overlays of the type shown in the example of FIG. 9 shown

Transfer functions 400. However, those described in the various herein

described transfer functions have certain characteristics or characteristics that allow a particularly good determination of the result image. Such features of transfer functions used will be described below. From the examples of the transfer functions 400 in FIG. 9, it is apparent that it is possible to form the spatial frequency transfer functions 400 within the aperture of the imaging optics 112 without local extremes, i. without local maxima or minima which would be smaller than the absolute extremes (i.e., the amplitudes of +1 and -1, respectively, in the example of FIG. This can be achieved by a monotone increasing or decreasing transfer function, or by a step function.

Such avoidance of local extreme values may in particular have advantageous effects with regard to the reduction of signal noise or artifacts in the resulting image. For example, those used by Tian and Waller

Transfer functions (see Tian and Waller: Figure 2, top left) local extreme values within the double aperture of the imaging optics on. Sometimes it can

occur that - for example, due to structural - there is a deviation between the actual aperture and the nominal aperture of the imaging optics. Then, the position of the local extreme values of the transfer function with respect to the actual aperture may be incorrectly positioned in the spatial frequency space; this causes mistakenly a frequency included in the images due to the local extreme values incorrectly positioned in the spatial frequency space with respect to the actual aperture strong reinforcement, which can lead to artifacts in the result image. In accordance with the various examples described herein, a transfer function having no local extremes within the detector aperture or double

Detector aperture is used, such an erroneous amplification of frequencies contained in the images due to a shifted positioned local extreme value of the transfer function can be avoided. It results in one

uniform propagation of the frequencies included in the acquired images.

From the in FIG. 9, it is further apparent that implementations are possible in which the spatial frequency transfer function within the aperture of the imaging optic 12, or within the double aperture of the imaging optic 112, assumes no values or substantially no values equal to zero, i. assumes only finite values other than zero. In general, it may sometimes be desirable to avoid that

Transfer function for spatial frequencies within the aperture of the imaging optics 1 12 or within the double aperture of the imaging optics 1 12 comparatively small values - for example, based on a maximum of all magnitude values of the

Transmission function for spatial frequencies within the corresponding range - assumes. For example, it would be possible for the transfer function for

Spatial frequencies within the aperture or the double aperture of the imaging optics 1 12 no magnitude values <5% of a maximum of all absolute values of the

Transfer function 400 for spatial frequencies within the aperture of the imaging optics 1 12, optionally no values <2%, further optional no values <0.5%. Such behavior may be e.g. be provided by a step function.

Such techniques are based on the knowledge that values equal zero for the

Transfer function 400 may correspond to a suppression of the corresponding frequencies contained in the images. Often, however, it may be desirable that within the aperture of the imaging optics 1 12 or within the double aperture of the imaging optics 1 12 no suppression of corresponding in the

Images included frequencies. For example, Tian and Waller: FIG. 2, top left, that in an extended area in the center of the aperture of the Optics, the transfer function can take values equal to zero. In this case, the smaller the ratio between the size of the aperture of the illumination module and the size of the aperture of the optics, the larger the areas in which the transfer function assumes values equal to zero. This means that, according to the reference techniques described in Tian and Waller, it may not be possible or only possible to a limited extent to determine a meaningful phase-contrast result image if the size of the aperture of the illumination module is significantly smaller than the size of the aperture of the optic. This limitation of the reference implementation may be remedied by the various techniques described herein. In particular, it may be possible by the appropriate choice of the transfer function without values equal to zero or very small values within the simple aperture of the imaging optics 1 12 or the double aperture of the imaging optics 1 12, even for comparatively large apertures of the imaging optics 1 12 or comparatively small apertures of the illumination module 1 1 1 to determine the result image with the phase contrast.

From the example of FIG. 9 it can also be seen that the figures shown there

Transmission functions for spatial frequencies outside the double aperture of the imaging optics 1 12 assume values equal to zero. In general, it may be possible to use transfer functions outside of that

Imaging optics 1 12 transmitted spatial frequencies assume values substantially equal to zero, ie typically outside the single aperture or the double aperture with partially phase-incoherent illumination. For example, it would be possible for the transfer functions used for spatial frequencies outside the single or double aperture of the imaging optics to have no magnitude values of> 5% of a maximum of all absolute values of the transfer functions for spatial frequencies within the single or double aperture of the imaging optics, optionally no values greater than 2%, further optional no values greater than 0.5%. In this way it can be avoided that artifacts or noise in the result image is amplified. FIG. 10 is a flowchart of an example method. First, a sample object is fixed in 1001, for example using a sample holder. For example, the sample object may be a phase object. For example, the sample object could Cells or cell cultures. The sample object could comprise a phase object. 1001 is optional.

Then, in 1002, the sample object is structured with one or more

Illuminated lighting geometries. This can be a corresponding

Lighting module to be controlled accordingly. For example, it would be possible for the sample object to be illuminated with two complementary illumination geometries, for example semicircular and different

Semicircles within the aperture of the corresponding lighting module

correspond.

In 1003, one or more images of the sample object are scanned using a

Imaging optics and by means of a detector, such as a CMOS or CCD sensor, detected. 1003 may include the appropriate driving of the detector. The image or images each include an image of the sample object. Different images are associated with different illumination geometries from 1002.

In some examples, two pairs of images may be detected, each associated with complementary semicircular illumination directions. In other examples, however, only two images or three images could be captured.

There could then be a difference, e.g. according to hinks ^ right

^ left ⁺ ^ right fopen ^" Junten

praise ⁺ back _{/ x}

where l (left) and l (right) denote the images respectively associated with a left or right oriented semicircular illumination geometry and where l (top) and I (below) denote the images respectively associated with a top or bottom oriented semicircular illumination geometry.

Also in Eq. 3, considering the denominator as normalization is optional. It would be possible to consider only the differences. It could also be the raw data l _left or I _right or I _above or I used _below , ie without pairing according to. Eq. 3 or Eq. Second

Then, in 1004, determining a result image, which is a

Has phase contrast. The determination of the result image is made in 1004 based on a transfer function which describes the imaging of the sample object by means of the corresponding optical system for the corresponding illumination geometries. The result image is also determined based on the at least one image captured in 1003. For this purpose, for example, a difference formation and, if necessary, standardization could be carried out beforehand from a plurality of images recorded in 1003, which with

different illumination geometries are associated.

For example, the method of FIG. 10 further comprises scaling a reference transfer function to a size of the aperture of the imaging optics. This means that an adaptation of the reference transfer function to the size of the aperture of the imaging optics can take place.

In summary, techniques have been described above to determine, even with comparatively large apertures of the imaging optics used, a result image which has a high contrast, which for example encodes the phase or the height of a sample object. These techniques are based on considering the size of the aperture of the imaging optics. In this case, for example, a predetermined reference transfer function can be scaled according to the size of the aperture of the imaging optics. The reference transfer function can therefore also be referred to as an artificial transfer function because it can have deviations from the theoretically expected transfer function due to the illumination geometry. Such techniques may have certain advantages. For example, the size of the aperture of the imaging optics can be flexibly dimensioned. In particular, for example, immersion objectives could be used. By means of the techniques described herein, by suitable choice of the transfer function, a particularly large phase contrast can be achieved in the resulting image. In particular, a gain of the phase contrast, for example, compared to the reference implementations according to Tian and Waller done. In addition, for example, it would be possible to digitally replicate certain forms of hardware-implemented phase-contrast images, such as Zernike contrast.

Of course, the features of the previously described embodiments and aspects of the invention may be combined. In particular, the features may be used not only in the described combinations but also in other combinations or per se, without departing from the scope of the invention.

The scaling of the amplitudes of the various ones described herein

Transfer functions is purely exemplary. For example, transfer functions having amplitudes of +1 and -1, respectively, have often been illustrated in the various examples described herein, however, in other examples, it may also be possible to use transfer functions of other amplitudes.

Further, for example, various implementations with respect to illumination of the sample object with partially coherent light have been described. The bandwidth of the transmitted spatial frequencies is equal to twice the aperture of

Imaging optics. In other examples, however, other techniques could be used for illumination, so the bandwidth of the transmitted

Location frequencies differently dimensioned. In the various examples described herein, this may be taken into account, for example by a corresponding scaling of a reference transfer function up to the theoretical maximum of the transmitted spatial frequencies. Further, various examples have been described herein in which a particularly large aperture of the imaging optics is used. The examples described herein can also be used for other cases, for example, in cases where the size of the aperture of the illumination module is greater than or approximately equal to the

Size of the aperture of the imaging optics is. Even in such cases, a result image can be achieved with a particularly strong contrast.

Claims

An optical system (100) comprising:

a sample holder (1 13) arranged to fix a sample object, an illumination module (1 1 1) arranged to illuminate the sample object with at least one structured illumination geometry (300),

- An imaging optics (1 12), which is adapted to an image of the illuminated with the at least one structured illumination geometry (300)

To generate a sample object on a detector (1 14),

the detector (1 14) arranged to detect at least one image of the sample object based on the image, and

a controller (1 15) arranged to be based on a

Transfer function (400) and the at least one image to determine a result image having a phase contrast,

wherein the transfer function (400) is one based on a size of a

Aperture of the imaging optics (1 12) scaled reference transfer function (400) corresponds.

2. Optical system (100) comprising:

a sample holder (1 13) arranged to fix a sample object,

a lighting module (1 1 1) arranged to illuminate the sample object with at least one structured illumination geometry (300),

- An imaging optics (1 12) which is adapted to an image of the illuminated with the at least one structured illumination geometry (300)

To generate a sample object on a detector (1 14),

a controller (1 15) arranged to be based on a

wherein a size of the aperture of the illumination module (1 1 1) is smaller than a size of the aperture of the imaging optics (1 12).

3. Optical system (100) according to claim 2,

wherein the transfer function (400) corresponds to a reference transfer function (400) scaled to a size of an aperture of the imaging optics (1 12).

4. Optical system (100) according to one of the preceding claims,

wherein the size of the aperture of the illumination module (1 1 1) is less than 50% of the size of the aperture of the imaging optics (1 12), optionally less than 20%, further optionally less than 5%. 5. Optical system (100) according to one of the preceding claims,

wherein the transmission function (400) for spatial frequencies within the single or double aperture of the imaging optics (1 12) has no magnitude values less than 5% of a maximum of all absolute values of the transmission function (400) for spatial frequencies within the single or double aperture of the imaging optics (1 12) , optionally no values less than 2%, further optional no values less than 0,

5%.

6. Optical system (100) according to one of the preceding claims,

wherein the transfer function (400) for spatial frequencies within the aperture of the imaging optics (1 12) has no local extreme values.

7. Optical system (100) according to one of the preceding claims,

wherein the transfer function (400) is determined as a function of the at least one structured illumination geometry (300).

8. An optical system (100) according to one of the preceding claims,

wherein the transfer function (400) is a step function.

9. Optical system (100) according to one of the preceding claims,

wherein the transfer function (400) is a monotone increasing or decreasing function, optionally a linear function or a sigmoid function.

10. Optical system (100) according to one of the preceding claims, wherein the transfer function (400) has an axis of symmetry (405) which corresponds to an axis of symmetry (305) of the at least one illumination geometry (300).

1 1. Optical system (100) according to one of the preceding claims,

wherein the transfer function (400) for spatial frequencies outside the single or double aperture of the imaging optics (1 12) does not have magnitude values greater than 5% of a maximum of all absolute values of the transfer function (400)

Spatial frequencies within the single or double aperture of the imaging optics (1 12), optionally no values greater than 2%, further optionally no values greater than 0.5%.

12. Optical system (100) according to one of the preceding claims,

wherein the controller (15) is arranged to determine the result image based on inverse Fourier transform Tichonov regularization.

13. A method comprising:

- Illuminating a sample object with at least one structured one

Illumination geometry (300),

- Generating an image of the at least one structured

Illumination geometry (300) illuminated sample object,

based on the image: capturing at least one image of the sample object, and

based on a transfer function (400) and the at least one image:

Determining a result image having a phase contrast,

14. The method according to claim 13,

the method of an optical system (100) according to any one of

Claims 1 - 12 is executed.