EP3186672A2

EP3186672A2 - Phase contrast imaging

Info

Publication number: EP3186672A2
Application number: EP15754213.5A
Authority: EP
Inventors: Lars STOPPE; Christoph HUSEMANN
Original assignee: Carl Zeiss Microscopy GmbH
Current assignee: Carl Zeiss Microscopy GmbH
Priority date: 2014-08-26
Filing date: 2015-08-25
Publication date: 2017-07-05
Also published as: WO2016030390A2; US20170276923A1; CN106716218A; WO2016030390A3; JP2017531201A; DE102014112242A1; US10338368B2

Abstract

The invention relates to an object (100) which is illuminated from at least one illuminating direction (110-1, 110-2). An intensity image of the object (100) is detected during the illumination for each illuminating direction (110-1, 110-2). A phase contrast image of the object (100) is generated on the basis of the at least one intensity image.

Description

Description Phase-contrast imaging

The present application relates to a method of generating a phase contrast image of an object and a corresponding optical device. In particular, the present application relates to techniques in which the object is illuminated sequentially from at least two illumination directions and the phase contrast image is generated based on corresponding intensity images.

In the optical imaging of objects, it may often be desirable to create a so-called phase contrast image. In a phase contrast image is at least a part of the

Image contrast caused by a phase shift of the light by the imaged object. In particular, those objects with comparatively higher contrast can be imaged, which cause no or only a slight weakening of the amplitude of the light, but a significant phase shift (phase objects). Typically, biological samples as an object in a microscope can have a greater phase change than

Cause amplitude change of the electromagnetic field.

Various phase contrast imaging techniques are known, such as the

Dark field illumination, oblique illumination, Differential Interference Contrast (DIC) or Zernike phase contrast. Other techniques would be e.g. the so-called schlieren method (English, knife edge) or the helical phase contrast.

Such aforementioned techniques have several disadvantages or limitations. So it is with the DIC technique, the Zernike technique, the Schlieren method and the helical

Phase contrast typically necessary to provide additional optical elements between sample and detector in the field of so-called detection optics over conventional amplitude imaging. This can lead to structural limitations, especially in modular microscopes. Costs are typically increased. For thin samples, darkfield illumination typically typically causes only a few photons to form, resulting in noisy, lower-quality images. A subsequent evaluation or analysis of the images can not or only partially possible be. An oblique illumination typically leads to an asymmetrical increase in contrast, which in turn can cause a reduced quality of the images.

Therefore, there is a need for improved phase-contrast imaging techniques.

In particular, there is a need for such techniques which enable a robust and simple construction of the optical device and which allow good image quality.

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

In one aspect, the present invention relates to a method of generating a phase contrast image of an object with an optical device. The method comprises sequentially illuminating the object from at least two illumination directions. The method further comprises detecting, for each illumination direction, an intensity image of the object during illumination from the respective illumination direction. The method further comprises combining output images to obtain a

Phase contrast image of the object. The source images are based on the detected

Intensity images. The at least two illumination directions are each assigned to a pair of illumination directions.

In other words, the at least two illumination directions can each form pairs or be arranged in pairs. It may be possible that a lighting direction is always associated with only one pair. However, it would also be possible for at least some of the illumination directions to be associated with a plurality of pairs.

For example, the optical device may be a microscopy device. Thus, the phase contrast image can enlarge the object. In particular, the at least two illumination directions may include different angles with the axis of the optical device along which an idealized light beam undergoes little or no deflection (optical axis). For example, in a simple implementation, the intensity images of the object may be combined from two or three or more arbitrary directions of illumination that include a different angle with the optical axis. Then, a certain phase contrast component in the phase contrast image can already arise. ). While the intensity images typically have no or no significant phase contrast, the phase contrast image can be generated by combining the output images which has a significant phase contrast component. In particular, in comparison with other conventional techniques of phase-contrast imaging, with the presently described method, the effect of a particularly simple implementation of the

Phase contrast imaging on the one hand, and the effect of a particularly simple and thus inexpensive and robust design of the optical device can be achieved.

In particular, it may be possible to use various conventional microscopy devices according to the technique described herein, without the need for modification of a detection optics. There may be different criteria for assigning two directions of illumination to a pair. For example, For example, geometric criteria of illumination directions of a pair may apply, such as with respect to the optical axis; such as e.g. be possible to produce a particularly high phase contrast component in the phase contrast image. For example, For example, the illumination directions of a pair could be symmetrical with respect to the optical axis and / or a plane including the optical axis. It would be alternatively or additionally possible that as an alternative or additional criterion of

Belonging to two lighting directions to a pair of a time of lighting and detecting is taken into account; e.g. those illumination directions can form a pair for which the respective intensity image is detected immediately or in rapid succession; such could e.g. a certain robustness against movement artifacts can be achieved. In general, as an alternative or additional criterion for the association of two illumination directions with a pair, a subsequent evaluation for generating the phase contrast image can also be taken into account; e.g. For example, for the two intensity images of a pair, a single output image could always be generated by combining these intensity images.

It would be possible for the two illumination directions of a pair to include correlating angles with the optical axis. Correlating angles can be e.g. mean substantially equal angles or substantially equal in terms of magnitude angles; In particular, with regard to technical limitations on accuracy, such as e.g.

systematic or statistical errors in detecting the intensity images by the optical device and / or structurally related Limitation a lighting device of the optical device to be characterized. If absolutely different angles are implemented but, for example, equal angles within the accuracy of the optical device, this can fulfill such a criterion of essentially the same angle. Such criteria apply below corresponding indications of angles and / or other properties of

Lighting directions or the optical device.

To describe geometric properties of the illumination directions, it may be helpful to describe the illumination directions by means of an illumination vector. The illumination vectors may be defined with respect to an origin of the optical device, such as with respect to the object and / or an intersection of a focal plane with the optical axis. A length of the illumination vectors may e.g. an amplitude of

Lighting from the respective lighting direction correspond; at the following

In discussing the orientation of different illumination vectors, it may be unnecessary to consider a length of the illumination vectors. The angle of the one

Includes illumination vector with the optical axis, then may correspond to the angle of the respective illumination direction. For example, it may be desirable that illumination vectors of a pair of

Illumination directions include an angle with each other, which is greater than 10 °, preferably greater than 20 °, more preferably greater than 40 °. It would alternatively or additionally also be possible for illumination vectors of a pair of

Lighting directions each include an angle with the optical axis, which is greater than 5 °, preferably greater than 10 °, more preferably greater than 20 °. It can thus be achieved that a difference vector between the two illumination vectors of a pair of illumination directions has a significant component perpendicular to the optical axis; this can particularly increase the phase contrast in the phase contrast image. In particular, it may be possible that the illumination vectors of two

Illumination directions of a pair of illumination directions by rotation about the optical axis of the optical device by an angle greater than 25 °, preferably greater than 50 °, particularly preferably greater than 85 ° into each other can be transferred. As a result, the difference vector becomes particularly large.

The two illumination directions of a pair of illumination directions may also be arranged such that associated illumination vectors include by rotation about the optical axis by an angle of 160 ° to 200 °, advantageously 175 ° to 185 °, most advantageously 180 ° with each other. It would also be possible that the associated

Illumination vectors by rotation about the optical axis by an angle of 70 ° to 1 10 °, preferably from 85 ° to 95 °, particularly advantageous of 90 ° into each other can. In other words, the two illumination vectors of a pair of illumination directions may lie in a plane and be arranged symmetrically or substantially symmetrically with respect to the optical axis. The optical axis may lie in this plane (be included by this plane), for example, when a rotation through 180 °, the two illumination vectors into each other. Thus, a comparatively large phase contrast component can be obtained in the phase contrast image, because the two

Lighting directions of a pair are arranged so complementary to each other.

In general, it may be desirable to use a larger number of illumination directions to obtain the phase contrast image. In particular, with an appropriate arrangement of the different illumination directions, the phase contrast component in the phase contrast image may increase. For example, it would be possible to have multiple pairs of

Lighting directions to consider. For example, it would be possible to sequentially select the object from 2 or 4 or 6 or 8 lighting directions or more

Illuminate lighting directions. For example, it would be possible for a first pair of illumination directions to include a first difference vector thereof

Lighting vectors determined. A second pair of illumination directions may correspond to a second difference vector of associated illumination vectors

determine. The first and second difference vectors may be at an angle to each other

include, e.g. an angle of 70 ° to 1 10 °, preferably from 85 ° to 95 °, particularly advantageously of 90 °.

It would accordingly also be possible for a first plane to be defined by the illumination vectors of a first pair of illumination directions. A second level may e.g. be defined by the illumination vectors of a second pair of illumination directions. The first level and the second level may e.g. enclose an angle of 70 ° to 1 10 ° with each other, preferably from 85 ° to 95 °, particularly advantageously from 90 °. The planes can e.g. be defined by the fact that the respective illumination vectors lie in the plane. It would also be possible for the planes to be defined by a normal vector oriented parallel to a difference vector of the respective illumination vectors; the optical axis can lie in the plane.

Thus, difference vectors of the illumination vectors of the two pairs of

Lighting directions thus include a comparatively large angle of up to 90 ° with each other; This allows the phase contrast in the phase contrast image along

different image directions are increased. For example, a phase contrast component in the Phase contrast image to be particularly large along such image directions, for the

Illumination vectors of a pair of illumination directions have a component perpendicular to the optical axis. In particular, a phase contrast component in the

Phase contrast image to be particularly large along such directions for which the difference vector of the illumination vectors of a pair of illumination directions has a component perpendicular to the optical axis. Therefore, it may be desirable to use complementary and / or symmetrically arranged illumination directions. In order to produce an isotropic phase contrast in the phase contrast image, it may be desirable that the illumination directions include evenly distributed angles with the optical axis.

It would be e.g. possible to generate multiple phase contrast images, the one

have direction-dependent phase contrast. For example, For example, a first phase contrast image could have a significant phase contrast along a first image direction and a second phase contrast image could have a significant phase contrast along a second phase direction

Image direction, wherein the first and second image directions are at an angle, e.g. an angle of 70 ° to 1 10 °, preferably from 85 ° to 95 °, particularly advantageously of 90 °. For example, For example, the method of generating the phase contrast image according to the present aspect may be applied to a first pair of illumination directions and applied to a second pair of illumination directions, thereby producing the first phase contrast image and the second phase contrast image. Difference vectors of the respective

Illumination vectors of the first pair and the second pair may include an angle with each other, e.g. an angle of 70 ° to 1 10 °, preferably from 85 ° to 95 °, particularly advantageously of 90 °. It can then be achieved that the first phase contrast image has a significant phase contrast along another image direction than the second phase contrast image. A direction-dependent phase contrast can be generated. This can enable a particularly high information content in a subsequent evaluation or analysis of the phase contrast images.

Accordingly, it would be possible for illumination vectors of a first pair of

Illumination directions by reflection at a first level, which includes the optical axis, can be converted into each other. Illumination vectors of a second pair of illumination directions may be merged by mirroring on a second plane including the optical axis and perpendicular to the first plane. This may mean that difference vectors of illumination vectors of a pair of illumination direction enclose an angle of substantially 90 ° with each other. Then, the phase contrast image may have a high phase contrast along different directions.

Above were various geometric arrangements of at least two

Called lighting directions. Basically, a variety of

Lighting devices are used to perform the lighting of the object with the different directions of illumination. For example, a scan mirror may be used e.g. be used in a field stop plane of the lighting device. It could also be an adaptive component in an aperture stop or illumination pupil of the

Lighting device used; e.g. the lighting device according to German Patent Application 10 2014 101 219.4 could be used. It would also be possible for the lighting device to comprise a light-emitting diode array. For example, can they

Light-emitting diodes of the light-emitting diode arrays can be arranged according to a Cartesian grid, wherein unit cells of the grid can be configured square or rectangular. Other rasters may also be implemented, such as hexagonal rasters, etc. It would be e.g. also possible that the light emitting diode array one or more rings of light emitting diodes, e.g. includes with different radii. Then, e.g. by driving a particular light emitting diode of the light emitting diode array, which has a certain distance from the optical axis, a particular illumination direction can be implemented.

Thus, the method may e.g. for each pair of illumination directions comprises: driving a first light emitting diode of the light emitting diode array to illuminate the object from a first illumination direction; and driving a second light-emitting diode of the LED array for illuminating the object from a second illumination direction. The

Light-emitting diode array can e.g. n have rows of LEDs and have m columns of LEDs. The first light-emitting diode can correspond to the ij light-emitting diode of the light-emitting diode array; Correspondingly, the second light-emitting diode can be selected from the following group: n-i + 1; j light-emitting diode; i; m-j + 1 light emitting diode; n-i + 1; m-j + 1 LED. i can thus denote a row index of the light-emitting diode array and thus be selected in the range from 1 to n, j can thus designate a column index of the light-emitting diode array and thus be selected in the range from 1 to m. Unless it is a square one

Light-emitting diode array, m = n. By such a symmetrical pairwise driving of the light-emitting diodes, in particular, the particular geometric relationship discussed above can be implemented: In the case of the selection of the second light-emitting diode according to n-i + 1; j or i; m-j + 1 may result in a scenario as described above in relation to the third level. In the case of the choice of a second light emitting diode according to i; m-j + 1 and a third light-emitting diode according to n-i + 1; j, the case of the third and fourth levels described above may arise. In the case of the selection of the second light-emitting diode according to n-i + 1; m-j + 1, the above-described case of the transfer of the illumination directions by rotation about the optical axis may result by an angle of substantially 180 °.

As explained above, in general, a different number and

Arrangement of lighting directions can be selected. In particular, pairs of illumination directions can be selected that are in a certain geometric relationship to each other. In such a case, in general, a pair may increase a phase contrast of structure of the lines in the phase contrast image perpendicular to a connection line within the pair, i. parallel to the difference vector; the connecting line within the pair may in this case correspond to a change in the difference of the illumination vectors. In general, a particularly rapid implementation of the method for generating the phase contrast image may be desirable; this can typically already be achieved with one or two pairs of illumination directions; if e.g. two pairs of

Lighting directions are used, the two pairs can be rotated by 90 °. In general, a more even increase in phase contrast can be produced in the

Phase contrast image can be achieved with a higher sampling rate of the space of the illumination directions; that is, in general, a larger number of pairs of illumination directions, e.g. evenly or statistically distributed over the corresponding space of lighting directions may be desirable. Different oblique illumination directions can in particular increase the plasticity of the phase contrast image. While in particular those illumination directions have been discussed above that include a finite angle with the optical axis, in the

In general, that illumination direction that is parallel to the optical axis can be used. Besides the influence of a number of considered

Illumination directions or intensity images used on the phase contrast of the phase contrast image, can also in terms of a signal-to-noise ratio, the

Using a larger number of lighting directions should be desirable. in the

In general, the more phase-contrast images are used to obtain the phase-contrast image, the less the phase contrast image will be. Signal noise can eg by Photon noise and / or camera noise of the individual intensity images are caused.

While techniques have been described above primarily relating to the type and / or arrangement of the illumination directions, techniques are described below primarily for generating the phase contrast image based on the detected

Affect intensity images.

In a simple implementation, the source images may correspond to the intensity images. The source images can also be applied by applying certain

Post-processing steps are obtained from the intensity images; thereby it may e.g. be possible to produce the phase contrast image with a comparatively high phase contrast and / or comparatively high signal-to-noise ratio. For example, the output images may be obtained by applying an operator to the acquired intensity images. The operator can e.g. selected from the following group: Amount; Square; Root; Sign inversion; Smoothing of pixels; Aberration correction of pixels; and normalizing to an average of pixel values of the respective one

Intensity image. For example, normalizing to the average may include subtracting an average of pixel values of the respective intensity image from each pixel value of the respective intensity image to obtain a corresponding output image.

In general, it is possible that an output image is determined for each intensity image, that is to say a 1: 1 assignment between intensity images and output images. But it would also be possible that several intensity images are combined to form an output image. For example, Thus, the output image can be determined by combining multiple intensity images

It would be e.g. possible that the method further comprises: combining those

Intensity images corresponding to a pair of illumination directions, each to an output image. In particular, in the event that the illumination directions associated with a pair meet certain geometric relationships, such a particularly high phase contrast proportion can be obtained in the generated phase contrast image. The thus obtained output images can then be combined again to produce the

Phase contrast image to produce. By combining two intensity images of a pair of illumination directions into a respective one output image, a particularly great flexibility in the process of combining can be ensured. In particular, it may be possible, for example, in the

Combine individual intensity images stronger or weaker to weight. Thus, e.g. certain optical effects can be compensated, which can occur by an angle which includes the respective illumination direction with the optical axis. For example, For example, an average intensity of an intensity image detected at a larger (smaller) angle included by the respective illumination direction with the optical axis may be smaller (larger). It may be possible to detect such effects through appropriate techniques

To balance. In particular, compared to techniques in which a larger number of intensity images and / or output images are combined at once, increased flexibility can be achieved.

For example, Combining the intensity images could be a subtraction of the respective ones

Comprise intensity images for obtaining the output images. Combining the

Output pictures may be an addition of the amounts of the output pictures to produce the

Phase contrast image include.

In general, therefore, the technique of combining is not particularly limited. The different output images may e.g. subtracted or added; The same applies to the intensity images, if they are combined to obtain an output image.

For example, combining the output images may include a weighted summation of the output images. In the weighted summation, each output image may be assigned a weighting factor. The method may e.g. for each output image, further comprising: determining the weighting factor based on an angle that the

corresponding illumination direction with the optical axis of the optical device includes. For example, the larger (smaller) the angle of the corresponding illumination direction with the optical axis, the larger (smaller) the weighting factor can be determined. Namely, a signal-to-noise ratio of the respective intensity image can typically be smaller (larger) for larger (smaller) angles of the respective illumination direction with the optical axis - with the amplitude of the corresponding illumination field remaining the same. For example, for a lower signal-to-noise ratio in the intensity images for larger angles of the illumination direction can be compensated. For example, the weighting factor may be determined such that a component of the respective illumination vectors is parallel to the optical axis assumes a predetermined length, for example, the same length for the different directions of illumination.

By performing a weighted summation, it may thus be possible to

different influences of the different illumination directions or the different pairs of illumination directions on the respective intensity images in the combination to take into account. That way it can be possible to have a particularly high quality

Phase contrast image - e.g. in particular compared to techniques which combine flatly different lighting directions to the right and left of the optical axis.

By means of the above techniques, it may be dispensable to use other optical elements, e.g. used in conventional phase-contrast imaging techniques. In particular, e.g. the illumination with light is such that the light in the optical path of the optical device between the object and a detector does not pass through elements selected from the group: polarizing filter; Prism; Wollaston prism; Phase ring; Gray filter. In particular, to established prior art techniques of phase-contrast imaging, e.g. DIC technology or the Zernike technique, so a simplified structure of the optical device can be achieved. This can reduce operating costs or reduce the susceptibility of the operation to errors. Furthermore, it may be unnecessary for the present method, particularly coherent light and / or

to use monochromatic light. For example, illumination can be done with incoherent light. In particular, the illumination of the object can also be done with white light, which has a significant spectral width. It may also be possible to design the illumination device of the optical device comparatively inexpensively and robustly against errors.

In particular, it may be dispensable within the detection optics, i. between object and detector to make modifications to the conventional intensity imaging. This may allow to use techniques described above in various microscopes. In particular, in comparison to the DIC technique, the Zernike technique and other techniques that interfere with the detection optics, such as. the streaking method or the helical one

Phase contrast, this has the effect of increased flexibility in use. Furthermore, by combining the phase intensity images to produce the phase contrast image, it can be ensured that even thin samples appear bright in the phase contrast image - this is particularly advantageous in comparison to phase contrast imaging techniques that are known in the art Dark field illumination based. With a suitable choice of the illumination directions, as described above, it can further be ensured that the phase contrast in the generated phase contrast image is uniformly increased in different directions. In particular, in comparison to the conventional technique of oblique illumination such an improved phase contrast image can be generated. Furthermore, by means of the techniques described above, it is possible to choose a diaphragm of the illumination device equal to or substantially equal to a diaphragm of the detection device of the optical device; In particular, it may be dispensable that the illumination aperture is larger than the detection aperture. Such can, for example, in comparison with the

Dark field illumination, a simplified structure of the optical device can be obtained. By means of the techniques described above, it may also be possible to use a

perform quantitative phase reconstruction, in which a contrast of the generated

Phase contrast image is proportional to a phase shift. In particular, in comparison to conventional image space operating techniques, e.g. dark field illumination, oblique illumination, the DIC technique, the Zernike phase contrast, the Schlieren method or the helical phase contrast, such a particularly meaningful phase contrast image can be generated. Other techniques that instead - as in the present

Picture space - operate in spatial frequency space, such as Fourier ptychography typically requires more extensive and complex computational steps than the present techniques operating in image space. Therefore, especially in real time applications or applications where rapid generation of the phase contrast image is desirable, the application of the present technique may be particularly desirable.

In general, the combining of the output images or the generation of the

Output images from the intensity images flexibly adaptable. Thus, e.g. the

Weighting factors in a weighted summation of the output images e.g. be customized or customized by a user. Thus, e.g. an optimized phase contrast of the generated phase contrast image can be made possible for a particular examined sample; in particular, this may be possible without having to undertake a hardware intervention in the optical device. In particular, this can provide a simplified adaptation to the conventional phase contrast imaging techniques

Allow imaging parameters to generate the phase contrast image; In particular, such imaging parameters can be adapted more robustly and safely. It may also be possible to adjust the imaging parameters retrospectively, ie after the illumination and detection of the object. This can increase flexibility in imaging. Furthermore, amplitude information of the imaged object is taken into account in the result image. This can especially in comparison to the prior art dark field illumination an advantage.

In another aspect, the present application relates to an optical device. The optical device is arranged to generate a phase contrast image of an object. The optical device comprises a lighting device that is set up to illuminate the object from at least two illumination directions. The at least two illumination directions are each assigned to a pair of illumination directions. The optical device includes a detector configured to be for each

Illumination direction to capture an intensity image of the object during illumination from the respective illumination direction. The optical device further comprises an arithmetic unit configured to be based on the acquired intensity images

Combining output images in the image space to produce a phase contrast image of the object.

The lighting device may comprise a light-emitting diode array. The light-emitting diodes of the light-emitting diode array can illuminate the object with incoherent light.

The optical device according to the presently discussed aspect of the application may be arranged to perform the method of generating a phase contrast image according to another aspect of the present application.

For such an optical device, effects comparable to the effects that can be obtained for the method of producing a phase contrast image with another aspect of the present invention can be achieved.

According to a further aspect, the present application relates to a method for generating a phase contrast image of an object with an optical device. The method includes illuminating the object from a direction of illumination. The illumination direction includes a finite angle with the optical axis of the optical device. The method further comprises capturing an intensity image of the object during the

Illuminate from the direction of illumination. The method further comprises processing the intensity image to produce the phase contrast image. Processing the

Intensity image includes rescaling the phase contrast image. It may thus be possible to generate a phase contrast image based on only a single intensity image. By means of such techniques it may be possible to

Phase contrast image to produce particularly fast. This can e.g. Have advantages in terms of moving samples. It may also be possible to illuminate the object with comparatively low light exposure. Furthermore, such a technique can reduce the effect

Have computational capacity when generating the phase contrast image. So it can be e.g.

be dispensable to process multiple intensity images or a larger number of intensity images. In this way, the generation of the phase contrast image by the post-processing can be done very quickly.

It would also be possible for the method to further include:

repeatedly performing the detection and processing of the intensity image for different directions of illumination and combining the thus generated

Phase contrast images. Thus, e.g. a particularly high phase-contrast component can be generated in the phase-contrast image. It can also be achieved that a signal-to-noise ratio in the thus obtained phase contrast image is particularly high.

In general, different rescaling techniques can be used. For example, rescaling may comprise: subtracting an average value of pixel values of the intensity image from pixel values of the intensity image. For example, can be the mean of all

Pixel values are each subtracted from each pixel value of the intensity image. The pixel values may be e.g. be proportional to a brightness of the object at the position represented by the respective pixel. In general, it is possible that the processing of the intensity image comprises further steps, e.g. Smoothing steps, etc. It would be e.g. possible that the processing of the

Intensity image continues to apply a magnitude operator to the rescaled

Intensity image includes. For example, For example, applying the magnitude operator may cause the respective positive pixel value to be output for each pixel.

According to a further aspect, the application relates to an optical device which

is arranged to produce a phase contrast image of an object. The optical device comprises a lighting device which is adapted to turn the object from a

Illumination direction to illuminate. The illumination direction includes a finite angle with the optical axis of the optical device. The illumination device further includes a detector configured to capture an intensity image of the object during the Illuminate from the direction of illumination. The optical device further comprises a computing unit configured to process the intensity image to produce the phase contrast image. The processing involves rescaling the

Phase contrast image.

For example, For example, the optical device according to the aspect currently discussed may be configured to perform a method of determining a phase contrast image according to another aspect of the present invention. For such an optical device, effects comparable to the effects that can be achieved for a method of determining a phase contrast image according to another aspect of the present invention can be achieved.

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. In particular, it may be possible to combine various features described above with respect to aspects in which the phase contrast image is generated based on one or more intensity images.

The above-described characteristics, features and advantages of this invention as well as the manner in which they are achieved will become clearer and more clearly understood

In connection with the following description of the exemplary embodiments, which in the

Connection with the drawings will be explained in more detail.

FIG. 1 illustrates, in two dimensions, the illumination of an object from two different illumination directions. Fig. 2A illustrates three-dimensionally illuminating an object of four

Lighting directions, with two lighting directions form a pair.

FIG. 2B illustrates three-dimensionally illuminating an object from three different directions of illumination, with two illumination directions each forming a pair. 3 schematically shows a light-emitting diode array that can be used to illuminate an object from different illumination directions.

4A illustrates a procedure for generating a phase contrast image from intensity images acquired under illumination of an object from different illumination directions, wherein an output image is generated for each intensity image, wherein the

Output images for generating the phase contrast image are combined.

FIG. 4B illustrates a procedure for generating a phase contrast image from intensity images acquired under illumination of an object from different illumination directions, the intensity images being combined in pairs into an output image, the output images being combined to produce the phase contrast image.

Fig. 5 schematically illustrates an optical device.

6 is a flowchart of a method for generating a phase contrast image.

Fig. 7 shows a dependency of weighting factors on an angle of corresponding illumination directions for the weighted summation of output images.

Fig. 8 illustrates illuminating the object from an illumination direction which includes a finite angle with the optical axis.

FIG. 9 illustrates pixel values of an intensity image captured for the illumination direction of FIG. 8. FIG.

Fig. 10 corresponds to Fig. 9, wherein the pixel values have been rescaled.

Fig. 11 corresponds to Fig. 10, wherein an absolute value operator has been applied to the pixel values.

Hereinafter, the present invention will be described with reference to preferred embodiments with reference to the drawings. In the figures, like

Reference signs 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, they are various elements shown in the figures reproduced so that their function and general purpose will be understood by those skilled in the art. Shown in the figures

Connections and couplings between functional units and elements can also be implemented as an indirect connection or coupling. A connection or coupling may be implemented by wire or wireless. Functional units can as

Hardware, software or a combination of hardware and software are implemented.

Hereinafter, techniques for generating a phase contrast image by means of an optical device will be explained. These techniques are based on processing or

Post-processing of one or more intensity images of an object, which are detected for specific lighting directions. In one scenario, the phase contrast image may be generated based on a single intensity image. In another scenario, based on multiple intensity images, the phase contrast image may be generated; by combining output images based on the intensity images, the phase contrast can be obtained in the phase contrast image. In such a scenario, the lighting directions can be arranged in pairs and in each case the

Intensity images are combined to form an output image that corresponds to a pair of

Lighting directions correspond. Subsequently, the thus obtained

Output images are combined to produce the phase contrast image.

The phase contrast of the phase contrast image can be determined by suitable choice of

Lighting directions are controlled. In particular, a geometric relationship of those illumination directions associated with a pair can provide a certain contribution to the phase contrast. For example, For example, two illumination directions of a pair of illumination directions may correlate with the optical axis of the optical angle

Include device. The correlating angle may mean, for example, that the two illumination directions of a pair are symmetrical with respect to the optical axis or in any case enclose equal angles with the latter in terms of magnitude. By means of such approaches, for example, a phase contrast that is isotropic or anisotropic over different image directions of the phase contrast image can be generated in a targeted manner. The phase contrast can also be determined quantitatively, as long as the illumination direction and possibly other parameters of the respective illumination fields, such as amplitude, etc., are known. In general, it may be desirable that two illumination directions of a pair of illumination directions include as large an angle as possible, eg, greater than 10 ° or greater than 20 ° or greater than 40 °. It may also be desirable that a first illumination direction of a pair of illumination directions includes a first angle with the optical axis, eg greater than 5 ° or greater than 10 ° or greater than 20 °. It may also be desirable that a second illumination direction of a pair of

Illuminating directions includes a second angle with the optical axis, e.g. is greater than 5 ° or greater than 10 ° or greater than 20 °. By such comparatively large angles between the illumination directions of a pair or between the

Lighting directions and the optical axis can be a comparatively large

Phase contrast component in the phase contrast image are obtained.

The present techniques thus allow by appropriate processing of the

Intensity images, i. after the actual capture, from the intensity images

Phase contrast images to produce. It is not necessary to provide further optical elements in the region of a detector of the optical device. In particular, illumination with light may be such that the light in the optical path of the optical device between the object and the detector does not pass through elements selected from the group consisting of: polarization filters; Prism; Wollaston prism; Phase ring; Gray filter. In particular, in comparison to established techniques of phase-contrast imaging, e.g. the DIC technique, such a simplified and particularly robust construction of the optical device can be achieved. The light can have a significant spectral bandwidth and does not have to meet a particular coherence criterion.

In Fig. 1, first, a one-dimensional view of a first illumination direction 1 10-1 and a second illumination direction 1 10-2 is shown. FIG. 1 also shows the optical axis 120 and the illuminated object 100. As can be seen from FIG. 1, the first illumination direction 1 10-1 includes a first angle 1 1 1 -1 with the optical axis 120. Furthermore, the second illumination direction 1 10-2 includes a second angle 1 1 1 -2 with the optical axis 120. In particular, the first illumination direction 1 10-1 and the second illumination direction 1 10-2 are arranged symmetrically with respect to the optical axis 120. The angles 1 1 1 -1, 1 1 1 -2 are equal in magnitude (if, starting from the optical axis 120, the value of the angle of zero is counted up, but, depending on the direction, provided with a sign).

For better geometric description of the illumination direction 1 10-1, 1 10-2 can be used on the illumination vectors. The illumination vectors may terminate in the object 100 and may include an angle with the optical axis 120 corresponding to the angle 1 1 1 -1, 1 1 1 -2 of the respective illumination device 1 10-1, 1 10-2. Such a scenario is shown in three dimensions in FIG. 2A. In Fig. 2A, 4 are

Illumination directions 1 10-1 - 1 10-4 represented by illumination vectors; the

Illumination directions 1 10-1 - 1 10-4 include equal angles with the optical axis 120. A first illumination direction 1 10-1 can be converted by rotation about the optical axis 120 by an angle of 180 ° into a third illumination direction 1 10-3.

Accordingly, the first illumination direction 1 10-1 and the third illumination direction 1 10-3, as well as the optical axis 120 are in a first plane 201 -1 (shown in dashed lines in Fig. 2A). The first illumination direction 1 10-1 and the third illumination direction 1 10-3 form a pair of illumination directions. For example, For example, associated intensity images of the first and third illumination directions 1 10-1, 1 10-3 may be combined to form an output image, e.g. by subtraction or addition of these intensity images.

It can also be seen from FIG. 2A that a second illumination direction 1 10-2 can be converted by a rotation about the optical axis 120 from 180 ° into a fourth illumination direction 1 10-4. The second illumination direction 1 10-2, the fourth illumination direction 1 10-4, and the optical axis 120 lie in a second plane 201 -2 (shown in dashed lines in FIG. 2A). For example, can be associated intensity images of the second and fourth

Lighting directions 1 10-2, 1 10-4 are combined to form an output image. The two output images thus obtained may in turn be combined to produce a phase contrast image, e.g. by subtraction or addition of these intensity images. The

Phase contrast image may e.g. along the x and y directions indicated in Fig. 2A.

The first plane 201 -1 of the first pair of illumination directions 1 10-1, 1 10-3 and the second plane 201 -2 of the second pair of illumination directions 1 10-2, 1 10-4 enclose an angle of 90 ° with each other. FIG. 2A also shows a connecting line or a first difference vector 1 15-1 of the illumination vectors of the first pair of illumination directions 1 10-1, 1 10-3. There is also a second difference vector 1 15-2 the

Illumination vectors of the second pair of illumination directions 1 10-2, 1 10-4 shown. It can be seen from FIG. 2A that these difference vectors 1 15-1, 15-2 also enclose an angle of 90 ° with each other, corresponding to the first and second planes 201 - 1, 201 - 2. Typically, the first pair of illumination directions increases 1 10-1, 1 10-3 den

Phase contrast in the phase contrast image perpendicular to the corresponding difference vector 1 15-

1 . Accordingly, the second pair of illumination directions 1 10-2, 1 10-4 increase the

2. Since in the scenario of FIG. 2A, the two difference vectors 1 15-1, 1 15-2 an angle of 90 ° with each other, ie evenly distributed over the space of the illumination directions 1 10-1 - 1 10-4, a comparatively isotropic phase contrast along the different image directions can be generated in the phase contrast image. It would be eg

possible, by taking into account further illumination directions (not shown in FIG. 2A) which include further angles with the optical axis 120, an increased isotropy of the

Phase contrast along different image directions of the phase contrast image to produce.

In the scenario of FIG. 2A, those directions along which there is a high phase contrast are tilted by 45 ° with respect to the xy image axis phase contrast image. By suitably forming pairs of illumination directions 1 10-1 - 1 10-4, it may also be possible to generate increased phase contrast along the xy image axes of the phase contrast image. This is shown in Fig. 2B. In the scenario of FIG. 2B, the illumination directions 1 10-1 and 1 10-2 form a first pair. The illumination directions 1 10-1, 1 10-4 form a second pair; this means that the second illumination direction 1 10-2 is associated with two pairs. It can also be seen from FIG. 2B that a third plane 201-3 (shown in dashed lines in FIG. 2B) includes the optical axis 120. Further, a fourth plane 201 - 4 (shown by dashed lines in Fig. 2B) includes the optical axis 120. The third plane 201 - 3 and the fourth plane 201 - 4 are oriented perpendicular to each other. The lighting vectors of the

Illumination directions 1 10-1, 1 10-2 of the first pair can be converted into one another by reflections of the third plane 201 -3. On the other hand, the illumination vectors 1 10 -1, 10 10-4 of the second pair of illumination directions can be converted into each other by reflections of the fourth plane 201 -4.

As can be seen from Fig. 2B, the difference vectors 1 15-1, 1 15-2 of the first and second pairs of illumination directions 1 10-1, 1 10-2, 1 10-4 are oriented perpendicularly at an angle of 90 ° to each other (See also Fig. 2A). At the same time, the difference vectors 1 15-1, 1 15-2 are each oriented parallel to the x, y image axes of the phase contrast image (unlike in FIG.

2A). Again, the intensity images corresponding to the illumination directions 1 10-1, 1 10-2,

1 10-4 of a pair, each combined into an output image. These

Output images can then be combined into a single phase contrast image.

This phase contrast image in turn has a comparatively isotropically distributed

Phase contrast along the different directions.

However, it would also be possible to generate a first phase contrast image for the first pair of illumination directions 1 10-1, 1 10-2 and to generate a second phase contrast image for the second pair of illumination directions 1 10-1, 1 10-4. These two phase contrast images then have an increased phase contrast along different image directions. This may be advantageous, for example, in the analysis of properties of the imaged object 100. In particular, the first phase contrast image, which is determined based on the first pair of illumination directions 1 10-1, 1 10-2, has an increased phase contrast along the y-image direction. Accordingly, the second phase contrast image, which is determined based on the second pair of illumination directions 1 10-1, 1 10-4, has an increased phase contrast along the x-image direction.

Highly symmetric cases were shown in FIGS. 2A and 2B, respectively. In Fig. 2A, it is possible to convert pair-forming illumination directions 1 10-1 - 1 10-4 by rotation about the optical axis by an angle of substantially 180 ° into each other. In Fig. 2B, it is possible to convert pair-forming illumination directions 1 10-1 - 1 10-4 by rotation about the optical axis 120 by an angle of substantially 90 ° into each other. For example, In detail with reference to FIG. 2B: Here, the first illumination direction 1 10-1 can be converted by rotation about the optical axis 120 by 90 ° in the second illumination direction 1 10-2 and by -90 ° in the fourth illumination direction 1 10 4th But it would also be possible to arrange the illumination directions such that they can be converted into each other by a different angle of rotation about the optical axis 120. So it would be possible for the couple to form

Illumination directions 1 10-1 - 1 10-4 by rotation about the optical axis 120 by an angle other than 90 ° or 180 °, e.g. an angle greater than 25 °, or preferably greater than 50 ° can be converted into each other. In general, the

Lighting vectors of a pair of lighting directions 1 10-1 - 1 10-4 by

Mirroring at the plane 201-3, 201 -4, which includes the optical axis 120 and has a normal vector parallel to the corresponding difference vector 1 15-1, 1 15-2 between the respective pairing illumination directions 1 10-1 - 1 10 4 is oriented, be converted into each other.

It is also generally not necessary that the illumination vectors of the

different directions of illumination 1 10-1 - 1 10-4 are arranged on a circle with respect to the optical axis 120, that can be converted by rotation about the optical axis 120 into each other as in the highly symmetrical case of Figs. 2A and 2B is the case. For example, The illumination directions can be 1 10-1 - 1 10-4 of a pair of

Illumination directions include different angles with the optical axis 120. Such geometric relationship as described with reference to FIGS. 2A and 2B can be implemented, for example, by various lighting devices which only one provide a finite number of possible illumination directions 1 10-1 - 1 10-4. In other words - depending on the lighting device - only very specific

Lighting directions 1 10-1 - 1 10-4 can be realized. Then, e.g. respectively the

nearest illumination direction 1 10-1 - 1 10-4 which has a given geometric criterion - e.g. Rotation about the optical axis 120 by a certain angle - as well as possible fulfilled. As part of the accuracy of the lighting device this selection then meets the geometric criterion.

In principle, it is therefore possible to use a wide variety of lighting devices 51 1

Illumination of the object from the different lighting directions 1 10-1 - 1 10-4 to use. FIG. 3 shows an example of a lighting device 51 1 in the form of a light-emitting diode (LED) array. The LED array 51 1 in FIG. 3 comprises 4 rows of LEDs and 4 columns of LEDs arranged symmetrically with respect to the optical axis 120. To z. B. lighting the object 100 along the direction of illumination to enable 10-1, the LED 300-1 1 can be controlled. In order to enable the illumination of the object 100 along the illumination direction 1 10-2, the LED 300-10 can be driven. In order to enable the illumination of the object 100 along the illumination direction 1 10-3, the LED 300-6 can be driven. In order to enable the illumination of the object 100 along the illumination direction 1 10-4, the LED 300-7 can be controlled. For example, analogous to a technique as illustrated in FIG. 2B, the following LEDs may correspond to 300-1-300-16 pairs of illumination directions 1 10-1 -1 10-4: LED 300-1 and LED 300-12 , LED 300-5 and LED 300-9; LED 300-2 and LED 300-14; LED 300-6 and LED 300-10; LED 300-2 and LED 300-15; LED 300-7 and LED 300-1 1; LED 300-4 and LED 300-16; LED 300-8 and LED 300-12. For example, the respective intensity images of a pair of illumination directions 1 10-1 - 1 10-4 may be subtracted to obtain an output image, respectively. The thus obtained output images may then be summed to produce a first phase contrast image. The first phase contrast image then has a particularly high phase contrast along the x image direction. Accordingly, it would also be possible to combine the following LEDs 300-1 -300-16 respectively into pairs of illumination directions 1 10-1 - 1 10-4 to produce a high phase contrast along the y-direction: LEDs 300-1 and LED 300-4; LED 300-5 and LED 300-8; LED 300-2 and LED 300-3; LED 300-6 and LED 300-7; LED 300-9 and LED 300-12; LED 300-13 and LED 300-16; LED 300-10 and LED 300-1 1; LED 300-14 and LED 300-15. In such a scenario, a first phase contrast image DPGC _X can be calculated according to the following formula: DPGCx = Ii - Ii3 + I5 - h + I2 - Iu + Ιβ - 110 + h - 115 + h - In + U - he + Is - 112

Accordingly, it is possible to have a second phase contrast image DPGC _y according to the following

To calculate equation:

DPGCy = Ii - + Is - Is + h - h + le - b + h - 112 + I13 - he + I10 - In + lu - 115 (2) where Ii is the intensity image with illumination of the object 100 from the respective

Lighting direction 1 10-1 - 1 10-14, which corresponds to the activation of the corresponding LED 300-1 - 300-16.

In Fig. 3, the LEDs 300-1 - 300-16 also have row and column indices noted; thus, e.g. the LED 300-7 has the row index i = 3 and the column index j = 2. In general, to to implement the illumination directions 1 10-1 - 1 10-4 according to one of FIGS. 2A, 2B, the method comprising: driving a first light-emitting diode 300-1 - 300-16 for illuminating the object from a first illumination direction 1 10-1 - 1 10-4 and driving a second LED 300-1 - 300-4 to illuminate the object from a second illumination direction 1 10-1 - 1 10-4. The first LED 300-1 - 300-16 then has the row index i and the column index j. The second LED 300-1 - 300-16 can then have the row index n-i + 1 and have the unchanged column index j, cf. Illumination directions 1 10-1, 1 10-2 in the scenario of FIG. 2B. The second LED 300-1 - 300-16 may also have the unchanged row index i and have the column index m-j + 1, cf. Illumination directions 1 10-1, 1 10-4 in the scenario of FIG. 2B. It is also possible that the second LED 300-1 - 300-16 the

Has row index n-i + 1 and the column index m-j + 1, cf. the couple forming

Illumination directions 1 10-1, 1 10-3, and the pair-forming illumination directions 1 10-2, 1 10-4 of Fig. 2A.

While a scenario in which a Cartesian grid of LEDs 300-1-300-16 was used is discussed in FIG. 3, generally, another arrangement of LEDs 300-1-100-16 may be used for the LED array 51 1 to use. For example, For example, it would be possible to use a circular array of LEDs 300-1 - 100-16 as the LED array 51 1. Then it would be e.g. in particular possible, the above with reference to Figs. 2A and 2B discussed

To implement scenarios particularly simply or particularly precisely, with which

different lighting directions 1 10-1 - 1 10-4 can be converted by rotation about the optical axis 120 into each other. In Fig. 4A, the combining of intensity images 401 -1 - 401 -6 is shown. In the scenario of FIG. 4A, three pairs 420-1-420-3, each consisting of two intensity images 401-1-401 -6, are obtained. For example, the first pair 420-1 could correspond to the intensity images 401 -1, 401 -2 of the illumination direction 1 10-1 and the illumination direction 1 10-3 (see Fig. 2A). In the scenario of FIG. 4A, a corresponding output image 402-1 - 402-6 is determined for each of the intensity images 401 -1 - 401 -6 (represented by the horizontal arrow in FIG. 4A). In a simple implementation, the output images 402-1 - 402-6 correspond to the intensity images 401 -1 - 401 -6. It can also be an operator on the captured

Intensity images 401 -1 - 401 -6 are applied to produce the output images 402-1 - 402-6. For example, the operator could be selected from the following group: Amount; Square; Root; Sign inversion; Smoothing of pixels; Aberrationkorrkektur of pixels and normalizing to an average of pixel values of the respective

Intensity image. Then, the output images 402-1 - 402-6 for generating the

Phase contrast image 410 combined. This combining can e.g. by summation,

Subtraction, division or multiplication of the different output images 402-1 - 402-6 happen to each other. Here, e.g. different output images 402-1 - 402-6 the different pairs 420-1 - 420-3 of intensity images 401 -1 - 401 -6 are given different signs; It would accordingly also be possible for a respective first output image 402-1 - 402-6 corresponding to a first intensity image 401 -1 - 401 -6 of a

Pairs 420-1-420-3, an inverse sign is obtained compared to a second output image 402-1 - 402-6. In combining the output images 402-1 - 402-6, weighting factors for the individual output images 402-1 - 402-6 can furthermore be used

be taken into account. Thereby, the influence of different output images 402-1 - 402-6 and thus different illumination directions 1 10-1 - 1 10-4 on the phase contrast image 410 can be controlled.

FIG. 4B shows another technique for generating the phase contrast image 410. In the scenario of Fig. 4B, two intensity images 401 -1 - 401 -6 of a pair 420-1 - 420-3 are combined into one output image 402-1 - 402-2, e.g. by addition,

Subtraction, multiplication or division. At the same time, it would be possible to apply one of the aforementioned operators to the different intensity images 401 -1 - 401 -6 or the thus-obtained output image 402-1 -402-3. Then, the output images 402-1 - 402-3 are in turn combined to produce the phase contrast image 410. While in Figs. 4A and 4B, a total of six intensity images 401 -1 - 401 -6 may be used, it may generally be possible to generate the phase contrast image 410 with only two intensity images 401 -1 - 401 -6. In Fig. 5, an optical device 500 is shown, with which the above

have implemented techniques described. The optical device 500 comprises a lighting device 51 1, e.g. in the form of the LED array (see Fig. 3) may be implemented. The object 100 is held by a sample holder 513 in an optical path of the light from the illumination device 51 1 to a detector 512. For example, can a

Transmission geometry can be implemented, in which the sample holder 513 is disposed between the lighting device 51 1 and the detector 512. It would also be possible to implement a reflection geometry. Furthermore, the optical device 500 has a computing unit 514 that may be configured to perform the various steps for

Generating the phase contrast image 410 (see Figures 4A and 4B). The optical device 500 may further include a memory 515, e.g. a non-volatile memory or volatile memory. The memory 515 may be corresponding

Control information for the computing unit 514 may be included to enable it to perform the various techniques for generating the phase contrast image as described above.

FIG. 6 shows a flow diagram of a method for generating the phase contrast image. The process starts with step S601. In step S601, the object 100 is illuminated. In particular, the illumination can be done with incoherent light. In particular, the light with which the object 100 is illuminated in step S601 may have a significant bandwidth, i. it can not be monochromatic. For example, the object 100 could be illuminated with white light in step S601.

In particular, the illumination of the object 100 takes place in step S601 from at least two illumination directions 1 10-1 - 1 10-4. The object is read in step S601 e.g. initially illuminated from a first illumination direction 1 10-1 - 1 10-4 and at the same time an intensity image 401 -1 - 401 -6 is detected. Then, the illumination of the object 100 takes place from a second one

Illumination direction 1 10-1 - 1 10-4; at the same time a further intensity image 401 -1 - 401 -6 is detected. In step S602, the intensity images 401 -1 - 401 -6 are combined to produce the phase contrast image 410. Various techniques may be used for combining become. In particular, when combining, weighting factors can be taken into account that also determine an influence of the different intensity images 401 -1 - 401 -6 or output images 402-1 - 402-6 based on them, which phase contrast image. In step S602, for example, the two intensity images 401 -1 - 401 -6 of a pair can each be combined to form an output image 402-1 - 402-6.

FIG. 7 shows an exemplary dependency of the influence of the different intensity images 401 -1 - 401 -6 on the phase contrast image 410 as a function of the angle 1 1 1, which the respective illumination direction 1 10-1 - 1 10-4 with the optical axis 120 includes, shown. For example, the larger (smaller) the larger the magnitude of the angle 1 1 1, the larger the influence of the respective intensity images 401 -1 - 401 -6.

FIG. 8 shows a scenario in which the object 100 is composed of a single object

Illumination direction 1 10-1 is illuminated. For this, e.g. a single LED 300-1 - 300-16 are turned on. From Fig. 8 it can be seen that the illumination direction 1 10-1 includes a finite angle 1 1 1 -1 with the optical axis 120. The object 100 in Fig. 8 has two stepped edges or rims and forms a plateau therebetween.

For the scenario of FIG. 8, a corresponding intensity image 401 -1 is detected, see FIG. 9. In FIG. 9, pixel values of the intensity image 401 -1 are shown in the x-z plane. It can be seen from FIG. 9 that at the edge of the object 100 shown on the left-hand side (right-hand side) in FIG. 8, the pixel values assume particularly large (small) values. In the area of the plateau of the object 100, the pixel values assume substantially constant values. Then, the intensity image 401 -1 is processed to generate the phase contrast image 410. FIG. 10 shows the rescaled intensity image 401 -1 and denotes output image 402-1. For this purpose, an average value of the pixel values of the intensity image 401 -1 is determined and this mean value is then subtracted from all pixel values. Therefore, in the illustration of Fig. 10, the curve of the pixel values shifts downward.

Then, an absolute value operator is applied to the output image 402-1, which is for each

Pixel value returns the amount. Furthermore, a smoothing can take place. This will do that

Phase contrast image 410 generated, see Fig. 1 1. On the basis of such techniques, the phase contrast image 410 can therefore be generated comparatively simply, for example, only on the basis of a single intensity image 401 -1 - 401 -6, which has a significant phase contrast component. Of course, it would optionally also be possible to have a plurality of phase contrast images 410 obtained in this way, for example for different ones

Illumination directions 1 10-1 - 1 10-4 then combine to form a result image, e.g. to improve the signal-to-noise ratio or to obtain an isotropic phase contrast component in the resulting image thus obtained. In such a scenario, it may be dispensable that the different directions of illumination 1 10-1 -1 10-4 are arranged in pairs.

In summary, techniques have been described above which include generating the phase contrast image by selectively combining intensity images obtained under

different lighting directions are detected. Furthermore, techniques have been illustrated that make it possible, by specific processing of an intensity image

Phase contrast image to produce. In this respect, e.g. be spoken of a digital phase gradient contrast. In particular, it may be unnecessary to use special optical elements, e.g. Prisms, etc., according to conventional techniques of phase-contrast imaging.

Although the invention has been further illustrated and described in detail by the preferred embodiments, the invention is not limited by the disclosed examples, and other variations can be derived therefrom by those skilled in the art without departing from the scope of the invention

To leave the scope of the invention.

Claims

Claims 1. Method for producing a phase contrast image (410) of an object (100) with an optical device (500),

the method comprising the following steps:

- sequentially illuminating the object (100) from at least two

Illumination directions (1 10-1 - 1 10-4),

for each illumination direction (1 10-1 - 1 10-4): acquisition of an intensity image (401 -1 -

401 -6) of the object (100) during illumination from the respective illumination direction (1 10-1 - 1 10-4),

wherein the at least two illumination directions (1 10-1 - 1 10-4) are each assigned to a pair (420-1 - 420-3) of illumination directions (1 10-1 - 1 10-4),

- Combining based on the acquired intensity images (401 -1 - 401 -6)

Output images (402-1 - 402-6) in the image space to produce a phase contrast image (410) of the object (100).

2. The method according to claim 1,

wherein the two illumination directions (1 10-1 - 1 10-4) of a pair (420-1 - 420-3) include correlating angles with the optical axis (120) of the optical device (500).

3. The method according to claim 1 or 2,

wherein illumination vectors of a pair of illumination directions (1 10-1 - 1 10-4) enclose an angle greater than 10 °, preferably greater than 20 °, more preferably greater than 40 °.

4. Method according to one of the preceding claims,

wherein illumination vectors of a pair of illumination directions (1 10-1 - 1 10-4) each include an angle with the optical axis (120) of the optical device (500) that is greater than 5 °, preferably greater than 10 ° preferably greater than 20 °. 5. The method according to any one of the preceding claims, wherein a first pair (420-1 - 420-3) of illumination directions (420-1 - 420-3) determines a first difference vector of associated illumination vectors,

wherein a second pair (420-1 - 420-3) of illumination directions (420-1 - 420-3) determines a second difference vector of associated illumination vectors,

wherein the first difference vector (1 15-1, 1 15-2) and the second difference vector (1 15-1,

1 15-2) enclose an angle with each other.

6. The method according to claim 5

wherein the method of generating the phase contrast image (410) for each of the first pair (420-1 - 420-3) and the second pair (420-1 - 420-3) of illumination directions (1 10-1 - 10-4 ) is applied to a first phase contrast image (410) and a second phase contrast image (410)

Phase contrast image (410) to produce

7. The method according to any one of the preceding claims,

wherein sequentially illuminating the object (100) from illumination directions (1 10-1 - 1 10-4) arranged in pairs (1 4-1 1 - 4 0-3) comprises:

for each pair (420-1 - 420-3): driving a first LED of a

Light-emitting diode arrays of a lighting device of the optical device (500) for illuminating the object (100) from a first illumination direction (1 10-1 - 1 10-4) and driving a second light emitting diode (300-1 - 300-16) of the light emitting diode Arrays for

Illuminating the object (100) from a second illumination direction (1 10-1 - 1 10-4),

wherein the light-emitting diode array has n rows and m columns,

wherein the first light emitting diode (300-1 - 300-16) corresponds to the i; j light emitting diode (300-1 - 300-16) of the light emitting diode array,

wherein the second light emitting diode (300-1 - 300-16) is selected from the group consisting of: n-i + 1; j light emitting diode (300-1 - 300-16); i; m-j + 1 LED (300-1 - 300-16); n-i + 1; m-j + 1 LED (300-1 - 300-16).

8. Method according to one of the preceding claims,

the method further comprising:

Combining those intensity images corresponding to a pair (420-1 - 420-3) of

Lighting directions (1 10-1 - 1 10-4) correspond to one output image.

9. Method according to one of the preceding claims,

the method further comprising: Applying an operator to the acquired intensity images to obtain the output images (402-1 - 402-6),

where the operator is selected from the following group: Amount; Square; Root; Sign inversion; Smoothing of pixels; Aberration correction of pixels; and normalizing to an average of pixel values of the respective intensity image (401 -1 - 401 - 6).

10. The method according to any one of the preceding claims

wherein combining the output images (402-1 - 402-6) comprises a weighted summation of the output images (402-1 - 402-6) having a weighting factor assigned to each output image,

the method optionally further comprising:

for each output image, determining the weighting factor based on an angle that the corresponding illumination direction (1 10-1 - 1 10-4) includes with the optical axis (120) of the optical device (500).

1 1. Method according to one of the preceding claims,

wherein illuminating the object (100) meets criteria selected from the group:

- Illuminate with incoherent light;

- Illuminate with white light; and

Illuminating such that light in the optical path of the optical device (500) between the object (100) and a detector does not pass through elements selected from the group: polarizing filter; Prism; Wollaston prism; Phase ring; and gray filters.

12. An optical device (500) arranged to generate a phase contrast image (410) of an object (100),

wherein the optical device (500) comprises:

a lighting device (51 1) which is set up to illuminate the object (100) from at least two illumination directions (1 10-1 - 1 10-4),

a detector (512) which is set up for each illumination direction (1 10-1 - 1 10-4) an intensity image (401 -1 - 401 -6) of the object (100) during illumination from the respective illumination direction (FIG. 1 10-1 - 1 10-4), an arithmetic unit (514) which is set up in the image space on the detected intensity images (401 -1-401 -6) based output images (402-1 - 402-6) for producing a phase contrast image (410) of the object (100) to combine.

13. An optical device (500) according to claim 12,

wherein the optical device (500) is arranged to perform a method according to any one of claims 1-11.

14. A method for generating a phase contrast image (410) of an object (100) with an optical device (500),

the method comprising the following steps:

- illuminating the object (100) from an illumination direction (1 10-1 - 1 10-4), wherein the illumination direction (1 10-1 - 1 10-4) includes a finite angle with the optical axis of the optical device (500) .

Detecting an intensity image (401 -1 - 401-6) of the object (100) during the

Illuminated from the illumination direction (1 10-1 - 1 10-4),

Processing the intensity image (401 -1 - 401 -6) to produce the phase contrast image (410),

wherein processing the intensity image (401 -1 - 401 -6) comprises rescaling the phase contrast image (410).

15. The method according to claim 14,

wherein rescaling comprises:

- subtracting an average of pixel values of the intensity image (401 -1 - 401 -6) from pixel values of the intensity image (401 -1 - 401 -6).

16. The method according to claim 14 or 15,

wherein processing the intensity image (401 -1 - 401 -6) further comprises:

Applying an absolute value operator to the rescaled intensity image (401 -1- 401 -6).

17. An optical device (500) arranged to generate a phase contrast image (410) of an object (100),

wherein the optical device (500) comprises:

a lighting device (51 1) arranged to illuminate the object (100) from an illumination direction (1 10-1 - 1 10-4),

wherein the direction of illumination (1 10-1 - 1 10-4) includes a finite angle with the optical axis of the optical device (500), a detector (512) arranged to detect an intensity image (401 -1 - 401 -6) of the object (100) during illumination from the illumination direction (1 10-1 - 1 10-4),

a computation unit (514) arranged to process the intensity image (401 -1 - 401 -6) for generating the phase contrast image (410),

18. An optical device (500) according to claim 17,

wherein the optical device (500) is further adapted to perform a method according to any one of claims 14-16