CN113302538A

CN113302538A - Variable angle illumination for phase contrast imaging with absorbing filters

Info

Publication number: CN113302538A
Application number: CN202080008311.6A
Authority: CN
Inventors: 拉尔斯·施托佩; 迪尔克·德林; 托马斯·米尔德
Original assignee: Carl Zeiss Microscopy GmbH
Current assignee: Carl Zeiss Microscopy GmbH
Priority date: 2019-01-09
Filing date: 2020-01-08
Publication date: 2021-08-24
Anticipated expiration: 2040-01-08
Also published as: CN113302538B; DE102019100419A1; WO2020144228A1

Abstract

The invention relates to a system (90) comprising a microscope (100) having: a lighting module (111); a sample holder (113); a detector (114); and an imaging optics unit (112) located between the sample holder 113) and the detector (114). The system also comprises at least one computing unit (115) designed to: the illumination module (111) is controlled to illuminate the sample object (390) with light from a plurality of illumination directions (700) 703, 381, 382), and the detector (114) is controlled to capture an image each corresponding to one of the plurality of illumination directions (700) 703, 381, 382). The system further comprises an absorption filter (800) located in the imaging optics unit (112) and having a position-dependent absorption rate (810).

Description

Variable angle illumination for phase contrast imaging with absorbing filters

Technical Field

Various examples of the invention generally relate to a system including a microscope and at least one computing unit. In this case, the microscope comprises an illumination module configured to illuminate the sample object with light from a plurality of illumination directions. Digital post-processing may be performed by means of the calculation unit to obtain a resulting image with a customized contrast. The invention relates in particular to the use of absorption filters arranged in the imaging optical unit of a microscope.

Background

In optical imaging of a sample object, it may often be worthwhile to generate a so-called phase contrast image of the sample object. In phase contrast images, at least some of the image contrast is caused by a phase shift of light passing through the sample object being imaged. In particular, this enables sample objects that cause no or only a small amplitude attenuation but a significant phase shift to be imaged with a relatively high contrast; such a sample object is also commonly referred to as a phase object. Biological samples as sample objects in microscopes can often cause phase changes that are relatively larger than electromagnetic field amplitude changes.

Various phase contrast imaging techniques are known, such as dark field illumination, oblique illumination, Differential Interference Contrast (DIC), or Zernike phase contrast.

Such techniques described above have various drawbacks or limitations. In general, it may be necessary to provide additional optical elements between the sample and the detector in the region of a so-called imaging optical unit in order to facilitate phase contrast imaging. This may result in structural limitations. Furthermore, there may be limitations in application: for example, fluorescence imaging may be made more difficult by providing additional optical elements.

Techniques are also known in which phase contrast can be obtained by means of so-called angularly variable illumination. In the present case, the angularly variable illumination associated with the present disclosure is intended to mean a technique in which a sample object may be illuminated with different illumination geometries over the entire area, i.e. in particular from different illumination directions. The illumination geometry is implemented by one or more illumination directions.

A first example of a technique by means of which images with phase contrast can be obtained by means of angularly variable illumination is disclosed in DE 102014112242 a 1. See also: tian, Lei and Laura Waller. "3D intensity and phase imaging from light field measurements in an LED array microscope [ 3D intensity and phase imaging obtained by light field measurement in LED array microscope ]. "optical 2.2(2015): 104-111. However, these techniques have certain limitations. For example, it has been observed that in these techniques, if the detector numerical aperture is larger than the illumination numerical aperture, the phase contrast intensity decreases. On the other hand, however, in the case associated with high resolution imaging, it may be worthwhile if a larger detector numerical aperture is used.

Disclosure of Invention

Accordingly, there is a need for improved phase contrast imaging techniques. In particular, there is a need for phase contrast imaging techniques that eliminate at least some of the above disadvantages and limitations.

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

In one example, a system includes a microscope. The microscope has an illumination module, a sample holder, and a detector. The microscope also has an imaging optical unit arranged between the detector and the sample holder. In addition, the system also includes at least one computing unit. In this case, the at least one calculation unit is configured to control the illumination module to illuminate the sample object with light from a plurality of illumination directions. In addition, the at least one calculation unit is further configured to control the detector in order to capture an image. In this case, each of the images corresponds to one of the plurality of illumination directions. The system also includes an absorption filter. The absorption filter is disposed in the imaging optical unit. The absorption filter has a position dependent absorption rate.

These illumination directions may implement different illumination geometries. In the present case, the illumination geometry can be formed in each case by one or more illumination directions.

Such a technique may also be referred to as angularly variable illumination accordingly, since different illumination geometries are used in each case for illuminating the entire area of the sample object or sample holder.

Due to the use of different illumination directions or different illumination geometries, the corresponding images may have different contrasts. In particular, in the case of phase objects, the images may have different contrasts. The resulting image with phase contrast can then be generated by combining the images.

As an example, in this case, the at least one computing unit may also be configured to combine the images with each other to obtain a resulting image. The resulting image may have a phase contrast. In the present case, depending on the type of combination, different phase-contrast may be obtained, including, for example, non-quantized phase-contrast or phase gradient contrast. Corresponding techniques are disclosed in particular in the following documents: DE 102014112242 a1 and DE 102017108873 a 1.

A particularly high image quality can be achieved by using an absorption filter. For example, in some examples, a particularly strong phase contrast may be formed, such as, for example, a phase gradient contrast. This means that even a small phase shift through the sample object can bring a large value to the phase contrast in the resulting image. The use of an absorption filter may additionally enable the size of the detector numerical aperture of the imaging optics to be determined in a flexible manner. In particular, a relatively large detector numerical aperture may be used. In particular, the detector numerical aperture may be selected to be larger than the illumination numerical aperture of the illumination module. In the present case, the use of a larger detector numerical aperture may lead to a high quality of the captured image and thus also of the resulting image.

A method includes controlling an illumination module of a microscope to illuminate sample objects on a sample holder of the microscope with light from a plurality of illumination directions. The method additionally includes controlling a detector of the microscope to capture images each corresponding to one of the plurality of illumination directions. An absorption filter having a position-dependent absorption is arranged in the imaging optics of the microscope.

A computer program or computer program product or computer readable storage medium comprising program code. The program code may be executable by a computing unit to perform a method. The method comprises controlling an illumination module of a microscope to illuminate a sample object on a sample holder of the microscope with light from a plurality of illumination directions. The method additionally includes controlling a detector of the microscope to capture images each corresponding to one of the plurality of illumination directions. An absorption filter having a position-dependent absorption is arranged in the imaging optics of the microscope.

Drawings

Fig. 1 schematically illustrates a system including a microscope and a computing unit, according to various examples.

FIG. 2 is a flow chart of an exemplary method.

Fig. 3 schematically illustrates an exemplary illumination module of a microscope configured for angularly variable illumination.

Fig. 4 schematically illustrates an illumination geometry that may be used in association with angularly variable illumination, according to various examples.

Fig. 5 schematically illustrates angularly variable illumination of a sample object without causing phase shift, according to various examples.

Fig. 6 schematically illustrates angularly variable illumination of a sample object causing a phase shift according to various examples.

Fig. 7 schematically illustrates angularly variable illumination of a sample object causing a phase shift, according to various examples, wherein in fig. 7 the detector numerical aperture is larger than the illumination numerical aperture.

Fig. 8 schematically illustrates angularly variable illumination of a sample object causing a phase shift, according to various examples, wherein in fig. 8 the detector numerical aperture is larger than the illumination numerical aperture, and wherein an absorption filter is used in the imaging optics of the microscope.

Fig. 9 schematically illustrates absorption rate profiles of absorption filters according to various examples.

Fig. 10 schematically illustrates absorption rate profiles of absorption filters according to various examples.

Detailed Description

The above described features, characteristics and advantages of the present invention and the manner of attaining them will become more apparent and the invention will become more clearly understood by reference to the following description of exemplary embodiments, which is to be construed in conjunction with the accompanying drawings.

In the drawings, like reference characters designate the same or similar elements. The drawings are schematic representations of various embodiments of the invention. The elements shown in the figures are not necessarily shown to scale. Rather, the various elements shown in the figures are presented so that their function and general purpose are readily apparent to those skilled in the art. Connections and couplings between functional units and elements shown in the figures may also be implemented as indirect connections or couplings. The connection or coupling may be implemented in a wired or wireless manner. The functional units may be implemented as hardware, software, or a combination of hardware and software.

Techniques for capturing images with the aid of a microscope are described below. In the present case, a microscope comprises an illumination module, a sample holder, imaging optics and a detector.

Techniques for determining a resulting image with customized contrast are described below. For example, the resulting image may image a phase object with a certain phase contrast. In the present case, the phase contrast does not necessarily have to be formed quantitatively, but can generally also be formed qualitatively. This means that the contrast does not have to reproduce the phase of the sample object one-to-one. In some examples, phase gradient contrast may be used, for example, i.e., contrast may indicate a phase change.

The techniques described herein may determine a resulting image by digitally post-processing one or more images of a sample object. For example, one or more images of the sample object may be intensity images that do not have phase contrast of their own.

One or more images of the sample object may be associated with different illumination geometries. This means that one or more images can be captured with the detector in each case while the sample object is illuminated with the corresponding illumination geometry. This is also referred to as angle variable illumination.

For example, different illumination geometries may be associated with different illumination directions. The different illumination geometries or the associated different images may be separated from each other by time-division multiplexing or frequency-division multiplexing. Separation by different polarizations is also possible. The illumination geometry may have a directional dependence. For example, the illumination geometry may have an illumination gradient along one or more spatial directions. For example, the illuminance may vary in a stepwise manner along the spatial direction, for example 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, or the like. The sample object may be a priori unknown; that is, the sample holder may hold different sample objects. The sample object may also be opaque to the light used. Depending on the type of sample object, it may be worthwhile to operate the illumination module and the detector using either a reflected light geometry or a transmitted light geometry.

According to various examples, absorption filters arranged in the imaging optical unit of the microscope are used in order to increase the intensity of the phase contrast. For this purpose, the absorption filter has a position-dependent absorption which varies with the lateral position perpendicular to the optical axis of the microscope.

Fig. 1 illustrates an exemplary optical system 90. By way of example, the optical system 90 may include a microscope 100, such as an optical microscope using a transmitted light geometry or using a reflected light geometry.

With the aid of the microscope 100, a measuring object or a small structure of a sample object fixed on the sample holder 113 can be represented in an enlarged manner.

To this end, the microscope 100 comprises an illumination module 111. The illumination module 111 may be configured to illuminate the sample holder 113 over the entire area with different illumination geometries in each case.

Whole area illumination may mean that there is no significant variation in illumination in the area of the sample object or sample holder 113. This distinguishes the techniques described herein from structured illumination having an illumination pattern.

The illumination module 111 has an illumination numerical aperture. The illumination numerical aperture defines an area from which light can be radiated onto the sample object.

In addition, the microscope 100 comprises an imaging optical unit 112 (sometimes also referred to as objective lens or detector optical unit) which is configured to generate an image of the measurement object on the detector area of the detector 114. The detector numerical aperture of the imaging optics 112 may, for example, facilitate bright field imaging and/or dark field imaging depending on the illumination geometry used.

In the example of fig. 1, the illumination module 111 is configured to facilitate angularly variable illumination of the measurement object. This means that different illumination geometries of the light for illuminating the measurement object can be realized by the illumination module 111. The different illumination geometries may each include one or more reflected illumination directions or illumination angles.

Here, in various examples described herein, different hardware implementations for providing different lighting geometries are possible. For example, the lighting module 111 may comprise a plurality of adjustable light sources (the light sources are not shown in fig. 1) configured to locally modify and/or generate light.

The computing unit 115 of the optical system 90 may control the illumination module 111 or the light source. The computing unit 115 may be implemented as a microprocessor or microcontroller, for example. Alternatively or additionally, the computing unit 115 may comprise, for example, an FPGA or an ASIC. Alternatively or additionally, the calculation unit 115 may also control or read the sample holder 113, the imaging optics unit 112 and/or the detector 114. The calculation unit 115 may also perform calculation operations in connection with digital post-processing of the images captured by means of the detector 114.

In some examples, the computing unit 115 may be integrated into the housing of the microscope 100. However, in other examples, the calculation unit 115 may also be arranged externally with respect to the microscope 100. The calculation unit 115 may be implemented by a corresponding computer program executed on a PC, for example.

FIG. 2 is a flow chart of an exemplary method. For example, the method according to fig. 2 may be performed by the calculation unit 115 of fig. 1. The method according to fig. 2 makes it possible to generate a resulting image with phase contrast by using angularly variable illumination.

First, in block 9001, the lighting modules 111 are controlled such that a plurality of lighting geometries are generated. This means that for example different light emitting diodes in the light emitting diode matrix can be switched on and off successively. A plurality of light-emitting diodes in the light-emitting diode matrix can also be switched on simultaneously in order to implement the illumination geometry in this way by means of simultaneous illumination from a plurality of illumination directions. For example, half-space lighting may be implemented, wherein all light emitting diodes of the half-space are switched on or off.

Block 9002 then involves controlling the detector 114 to capture a plurality of images. In this case, the controlling lighting modules 111 in block 9001 and the controlling detectors 114 in block 9002 are implemented in a synchronized manner such that the various captured images are each assigned to a corresponding lighting geometry or to one or more corresponding lighting directions forming the respective lighting geometry. Time division multiplexing or wavelength division multiplexing or polarization division multiplexing may be used.

In optional block 9003, the images captured in block 9002 are then combined with each other to obtain a resulting image in this way. The resulting image has improved contrast, e.g., phase contrast.

In block 9003, a variety of techniques may be used to combine the captured images with one another. The phase contrast may vary depending on the combination (e.g., with or without normalization, or with or without absolute value formation, etc.). Corresponding techniques are described, for example, in the following documents: DE 102014112242 a1 and DE 102017108873 a 1.

Optionally, in block 9003, image processing techniques may also be applied, for example, to the respective captured images and/or the resulting images. Examples of image processing techniques include: background normalization; noise suppression; frequency filtering; frequency manipulation; and the like.

The use of an absorption filter in the beam path makes it possible in particular to obtain a phase contrast which is formed particularly uniformly over all the relevant spatial frequencies of the phase. This will be explained in more detail below.

Fig. 3 illustrates an exemplary embodiment of a lighting module 111. Fig. 3 is an illustration of an illumination module 111 that extends perpendicular to an optical axis 309 in a lateral plane spanned by the X-axis and the Y-axis.

As is apparent from fig. 3, the lighting module 111 comprises a matrix of light emitting diodes 121-1, 121-2 (although other embodiments are possible). In this case, the light emitting diode 121-1 is arranged within the detector numerical aperture 319 of the imaging optical unit 112, and the light emitting diode 121-2 is arranged outside the detector numerical aperture 319 of the imaging optical unit 112. This means that, in particular, both bright-field imaging as well as dark-field imaging can be realized by means of the illumination module 111.

In various examples described herein, the detector numerical aperture 319 can be large enough such that all of the light emitting diodes can be disposed within the detector numerical aperture 319 of the imaging optics unit 112. In this case, dark-field imaging cannot be performed.

Fig. 4 illustrates aspects associated with the individual operation of the different light emitting diodes 121-1, 121-2 of the lighting module 111. Fig. 4 is a sectional view along the axis X-X' of fig. 3.

In the example of fig. 4, the illumination geometry 700 is realized by means of isolated switching on of the light-emitting diode 121-1 marked with an arrow. As a result, the sample object is illuminated over the entire area from a particular illumination direction corresponding to the relative arrangement of the labeled light emitting diodes 121-1 with respect to the optical axis 309. However, in general, each illumination geometry may also switch on more than one light emitting diode 121-1, 121-2, such that the corresponding illumination geometry consists of multiple illumination directions. A wide illumination area may also be used. In this case, the different illumination geometries may have at least partially different illumination directions.

The operation of the variable angle illumination for generating a resulting image with phase contrast is described below in conjunction with fig. 5-7.

Here, fig. 5 illustrates aspects of illumination of a sample object 390 disposed in a sample plane 302, where the sample object 390 does not cause a phase shift of incident light.

In fig. 5, the illumination numerical aperture 301 of the illumination module 111 is schematically shown as a field stop (again, the focal distance determines the illumination numerical aperture; however, for the sake of simplicity, the focal distance is not separately represented in fig. 5). Also shown is a diameter 305 of the exit pupil defining the illumination aperture 301. The diameter 305, together with the focal length, defines the numerical aperture.

In fig. 5, two illumination geometries 701 (solid lines) and 702 (dashed lines) are used. Corresponding

central illumination directions

381, 382 are also shown. The light of the illumination geometry 701 is incident on the sample object 390 from the upper half space; and the light of the illumination geometry 702 is incident on the sample object 390 from the lower half-space. An exemplary embodiment of the

illumination geometry

701, 702 may be realized, for example, by means of an illumination module 111 with a matrix of light emitting diodes 121-1, 121-2, wherein, in association with the illumination geometry 701, all light emitting diodes 121-1, 121-2 above the center line are switched on, and wherein, in association with the illumination geometry 702, all light emitting diodes 121-1, 121-2 below the center line are switched on (see also fig. 3, wherein the corresponding center lines are indicated by dash-dot lines).

In the example of fig. 5, the imaging optical unit 112 includes a

lens pair

311, 312, wherein a conjugate plane (pupil plane) is defined between the

lenses

311, 312. Also shown in fig. 5 is the diameter 315 of the entrance pupil of the imaging optical unit 112, which defines a detector numerical aperture 319 (in addition, the detector numerical aperture 319 is also determined by the focal length of the imaging optical unit 112, but for simplicity the focal length is not shown in fig. 5).

At the far right of fig. 5, the detector plane 321 is shown on which the light is focused. In addition, fig. 5 also shows an optical axis 309 (chief ray) (dotted line in fig. 5).

Since the sample object 390 in fig. 5 is an amplitude object and does not cause a phase shift of incident light, the direction of light is not changed when passing through the sample object 390. This is different in the scenario in fig. 6.

Fig. 6 illustrates aspects of illumination of a sample object 390. In the present case, the example in fig. 6 corresponds in principle to the example in fig. 5. However, in the example in fig. 6, a phase object is used as the sample object 390. In the example of fig. 6, the sample object causes the light to be deflected upward away from the optical axis 309. Thus, not all light may pass through the detector numerical aperture 319. In particular, a portion of the light (marked with an arrow) associated with the illumination geometry 702 is not imaged onto the detector plane 321 because it is not located within the detector numerical aperture 319. In contrast, all light associated with the illumination geometry 701 may be focused onto the detector plane 321.

Thus, there is an asymmetry between the images associated with the

illumination geometries

701, 702. The model qualitatively explains the presence of phase contrast in the resulting image obtained by combining images corresponding to the

illumination geometries

701, 702. The combination may be achieved by addition or difference formation.

For smaller phase gradients, the difference between the images corresponding to the

illumination geometries

701, 702 is equal or approximately equal to zero: this is because light from both illumination halves (top and bottom of fig. 6), although shifted due to the (small) phase gradient, can still pass through the system without being obstructed and impinging on the detector. This is particularly applicable where the detector numerical aperture 319 is relatively large and therefore light from both illumination halves is located within the detector numerical aperture 319. This finding is explained in connection with fig. 7.

Fig. 7 illustrates aspects of illumination of a sample object 390. In the present case, the example in fig. 7 corresponds in principle to the example in fig. 6. However, in the example of fig. 7, an enlarged detector numerical aperture 319 is used as compared to the example of fig. 6. In particular, in the example in fig. 7, the detector numerical aperture 319 (schematically illustrated by a larger diameter 315 than the diameter 305) is larger than the illumination numerical aperture 301.

In such a scene as shown in fig. 7, all light of the illumination geometry 702 also reaches the detector plane 321 (see in particular the rays marked with arrows in fig. 7 and 6).

Fig. 7 qualitatively explains why the size of the detector numerical aperture 319 has to be relatively small according to the reference embodiment, especially with respect to the illumination numerical aperture 301. A description of a technique that makes it possible to use a large detector numerical aperture 319 without losing the possibility of also obtaining a resulting image with phase contrast is given below. The use of a large detector numerical aperture 319 in principle makes it possible to capture images with high resolution.

Fig. 8 illustrates aspects of illumination of a sample object 390. In the present case, the example in fig. 8 corresponds in principle to the example in fig. 7. In particular, also in the example in fig. 8, the diameter 315 of the entrance pupil associated with the detector aperture 319 is larger than the diameter 305 of the exit pupil associated with the illumination aperture 301, as already explained above in connection with fig. 7. This is intended to schematically demonstrate that the detector numerical aperture 319 may be larger than the illumination numerical aperture 301 (where this consideration takes into account the focal lengths of the illumination and the detector optics, which are not represented in fig. 8 for simplicity reasons).

In the example in fig. 8, an absorption filter 800 is provided, which is arranged in the imaging optical unit 112 and has a position-dependent absorption (the position-dependent absorption is illustrated in fig. 8 by the non-solid lines of the absorption filter 800 oriented in the X direction).

By using such a position-dependent absorption filter 800 with absorption, it can be achieved that light at a greater lateral distance from the optical axis 309 is absorbed to a greater extent due to the phase shift of the sample object 390 in the region of the pupil plane of the imaging optical unit 112. This in turn makes it possible to obtain a resulting image with phase contrast even if a large detector numerical aperture 319 is used, by combining different images corresponding to

different illumination geometries

701, 702.

By appropriately dimensioning the position dependence of the absorption rate, it can also be achieved that an enhanced phase contrast can be obtained even for less different illumination geometries (e.g. by implementing the illumination geometry using light emitting diodes 121-1, 121-2 that are close together).

Details associated with the position dependence of the absorbance of the absorption filter 800 are described below in connection with fig. 9 and 10.

Fig. 9 and 10 illustrate aspects associated with an absorptive filter 800. In particular, fig. 9 and 10 show the position dependence of the absorption 810 of the absorption filter.

In the examples of fig. 9 and 10, the greater the distance from the optical axis 309 (i.e., in the lateral direction, in the X direction in fig. 5 to 8), the greater the value taken by the absorptance 810. In the present case, the absorption 810 has a stepped radial profile in the exemplary embodiment in fig. 9; and in the embodiment of fig. 10 has a gradual radial profile.

The position dependence of the absorption 810 can be embodied in a rotationally symmetric manner with respect to the optical axis 390 in the lateral plane (spanned by the X-axis and the Y-axis). However, non-rotationally symmetrical embodiments are also possible: in the examples of fig. 9 and 10, in particular, a monotonic profile of the absorbance 810 is shown. In some examples, the absorptivity 810 can also be embodied as locally non-monotonic (e.g., along the X-axis or along the Y-axis) as long as the integral over the region remains monotonic. It is always true here that the integral of the light over the area of the illumination geometry (in the XY plane, perpendicular to the optical axis 309) decreases monotonically with increasing distance from the optical axis 309.

The absorption 810 may increase monotonically as a function of distance from the optical axis 309.

Such an embodiment of the position dependence of the absorption 810 makes it possible to achieve the effect that the greater the lateral distance from the optical axis 309, the greater the attenuation of the light rays (as described above in connection with fig. 5 to 8), and thus the enhancement of the phase contrast in the resulting image.

The examples in fig. 9 and 10 additionally show an inner region 801 adjacent the optical axis 309, and an outer region 802 surrounding the inner region 801 in a manner offset in the lateral direction. The absorption 810 in the outer region 802 is greater than the absorption in the inner region 801. In this case, it was observed in various tests that a relatively large difference between the absorbance 810 in the outer region 802 and the absorbance 810 in the inner region 801 may be worthwhile. For example, the absorbance 810 in the inner region 801 may be no greater than 10%, and the absorbance in the outer region may be > 50%. It can thereby be achieved that the light passing through the imaging optical unit 112 (and in particular through the pupil plane in the vicinity of the optical axis 309) is not attenuated or not significantly attenuated, with the result that the image has a strong signal. On the other hand, by sizing the absorbance 810 in the outer region 802 to be greater than 0% (e.g., in the range of 10% to 50%), it can be achieved that the resulting image has phase contrast, but also has high resolution.

For example, where the absorbance 810 in the outer region 802 is sized to be in the range of 10% to 20%, it may also be combined with fluorescence imaging (typically with relatively low light intensity in this case). Too high an absorption can be adjusted by the tendency of the resolution to decrease.

In the pupil plane, rays at a larger distance from the optical axis 309 correspond to high spatial frequencies. The overall quality of the various captured images is not limited, or not significantly limited, due to the limited damping of high spatial frequencies caused by the absorption 810 sized as described above.

Fig. 9 and 10 additionally illustrate diameter 305 and diameter 315, respectively. This allows specifying a relatively quantitative sizing of the position dependence on the absorption rate 810. As is evident from fig. 9 and 10, for example, the size of the diameter of the inner region 801 is approximately equal to the size of the diameter 305. Generally, the diameter of the inner region 801 may be in the range of 80% to 120%, optionally in the range of 80% to 100%, of the diameter 305 of the lighting module 111. In addition, in conjunction with fig. 9 and 10, it can also be seen that the inner region 801 and the outer region 802 each have a smaller diameter than the diameter 315.

As described above, this makes it possible to make the size of the illumination numerical aperture 301 of the illumination module 111 smaller than the size of the detector numerical aperture 319 of the imaging optical unit 112; while nevertheless a resulting image with phase contrast can be obtained.

In summary, a description has been given above of a technique which makes it possible to transmit even small phase information (e.g. phase gradient information) by means of angularly variable illumination during illumination. A resulting image with a particularly high phase contrast can thus be obtained by the combination of the individual images. In the various techniques described herein, this is achieved by maximizing the difference between two images corresponding to different illumination geometries already with a small deflection of the phase gradient not equal to zero. This is achieved in particular by attenuating the rays of the phase object that are directed away from the optical axis. For this purpose, absorption filters are used which reduce the light intensity of the light. The absorption filter may be, for example, a gradient filter or an absorption filter embodied in a stepped manner. The use of an absorption filter results in a difference unequal to 0 even for small phase shifts. These differences are (at least piecewise) proportional to the gradient of the phase.

It goes without saying that the features of the embodiments and aspects of the invention described above can be combined with each other. In particular, the features can be used not only in the combination described but also in other combinations or on their own, without departing from the scope of the invention.

For example, a description has been given above of a technique of combining captured (raw) images to obtain a result image. In the present case, the captured image may typically be post-processed, for example by applying filters in the frequency domain. These digital filters may be selected depending on the absorption filter used. This is based on the insight that: in the case of weak absorption of the filter, only low spatial frequencies or small phase gradients can be transmitted weakly. A (digital) frequency filter may then be used which amplifies the frequency range of the one or more original images and/or the resulting image corresponding to the attenuation region. For example, the frequency range may be multiplied by a factor, while all frequencies outside the range remain unchanged.

Other conventional image processing techniques will also work.

Claims

1. A system (90) comprising:

a microscope (100) having an illumination module (111), a sample holder (113), an imaging optical unit (112) of a detector (114) arranged one stage between the sample holder (113) and the detector (114),

-at least one calculation unit (115) configured to: controlling the illumination module (111) to illuminate a sample object (390) on the sample holder (113) with light from a plurality of illumination directions (700) 703, 381, 382), and controlling the detector (114) to capture images each corresponding to one of the plurality of illumination directions (700) 703, 381, 382),

-an absorption filter (800) arranged in the imaging optics unit (112) and having a position dependent absorption (810).

2. The system (90) of claim 1,

wherein the absorption (810) takes a value that is larger the distance to the optical axis (309) of the microscope (100).

3. The system (90) of claim 1 or 2,

wherein the absorption rate (810) has a gradual or stepped radial profile.

4. The system (90) of any one of the preceding claims,

wherein the absorption filter (800) has an inner region (801) with a first absorption rate (810),

wherein the absorbing filter (800) has an outer region (802) surrounding the inner region and having a second absorption rate (810),

wherein the second absorption rate (810) is greater than the first absorption rate (810).

5. The system (90) of claim 4,

wherein the absorption (810) of the inner region (801) is not more than 10%, and/or

Wherein the outer region (802) has an absorption rate (810) greater than 50%.

6. The system (90) of claim 4 or 5,

wherein the diameter of the inner region (801) is in the range of 80% to 120% of the diameter (305) of the exit pupil, wherein the exit pupil is associated with the illumination aperture (301) of the illumination module (111).

7. The system (90) of any of claims 4 to 6,

wherein the inner region (801) and the outer region (802) each have a diameter smaller than a diameter (315) of an entrance pupil associated with a detector aperture (319) of the imaging optical unit (112).

8. The system (90) of any one of the preceding claims,

wherein the illumination numerical aperture (301) of the illumination module (111) is smaller than the detector numerical aperture (319) of the imaging optical unit (112).

9. The system (90) of any one of the preceding claims,

wherein the absorption filter (800) is arranged in a pupil plane of the imaging optical unit (112).

10. The system (90) of any one of the preceding claims,

wherein the at least one calculation unit (115) is further configured to combine the images with each other to obtain a result image, wherein the result image has a phase contrast.

11. A method, comprising:

-controlling an illumination module (111) of a microscope (100) for illuminating a sample object (390) on a sample holder (113) of the microscope (100) with light from a plurality of illumination directions (700) 703, 381, 382),

-controlling a detector (114) of the microscope (100) to capture images each corresponding to one of the plurality of illumination directions (700) 703, 381, 382),

wherein an absorption filter (800) having a position dependent absorption rate (810) is arranged in an imaging optical unit (112) of the microscope.

12. The method of claim 11, wherein the method is performed by a computing unit (115) of a system (90) of any of claims 1 to 10.