DIGITAL HOLOGRAPHIC MICROSCOPE WITH A MULTI-OFFSET INTERFEROMETER

Description

[0001] The invention relates to digital holographic microscopes and methods for extending the uniqueness range in measurements, in particular in holographic or interferometric measurements.

[0002] The present invention relates to a digital holographic microscope DHM and a method for capturing and processing an overall image of a measured object, which can also be referred to as a measurement object, in particular a cell structure or an object to be tested.

[0003] Traditionally, the application of a synthetic wavelength concept is based on an evaluation of images with two or more different wavelengths.

[0004] For effective 3D measurement of cells and cell agglomerates, a large depth measurement range is advantageous. A large depth measurement range is also beneficial when, in addition to cells, solids in fluids, such as body fluids and / or secretions, are to be measured. With a single-wavelength interferometer, the depth measurement range is limited to one wavelength. To increase the measurement range, a second, slightly different wavelength is often added. This results in two independent interferograms. The uniqueness range of the measurement for the combined synthetic wavelength lies at the least common multiple of the wavelengths divided by the difference between the wavelengths. This procedure is described by the term "synthetic wavelengths." WO 2010 / 122530 A1 shows a digital holographic microscope in the form of a lateral shear interferometer.

[0005] The object of the invention is to improve digital holographic microscopes (DHMs), and in particular associated image processing methods, in such a way as to improve the degree of uniqueness in measurements, especially holographic measurements. In particular, it is intended to obtain depth or thickness information of measured objects.

[0006] The problem is solved by a device according to the main claim and a method according to the dependent claim.

[0007] According to a first aspect, a digital holographic microscope is proposed for capturing and processing a first overall image of a measured object, measured with a first offset, with a device for generating at least one further overall image with at least one offset different from the first offset.

[0008] In an interferometer, the fringe pattern is created by the inclination of two beams relative to each other. If the beams contain plane waves, this results in an equidistant fringe pattern, with the fringe spacing depending on the angle of inclination of the two beams. In our case, the light is focused in the interferometer, with the focus located in the prismatic deflecting mirror. This focus is then the light source, which is collimated by the subsequent lens L2, resulting in nearly plane waves – which also carry object information. The lateral offset in the prismatic deflecting mirror allows the position of this light source to be shifted, which then influences or determines the inclination of the collimated beam.

[0009] According to a second aspect, a method is proposed for capturing an overall image containing superimposed information components of different imaging modes and measured using a digital holographic microscope, for example of a biological cell structure, a cell agglomerate, a medical sample, and / or solids, sediments and / or microcrystals in body fluids and / or secretions, in object space, with the steps of variably adjusting an offset, in particular by tilting a birefringent plate, carried out by means of a computer device.

[0010] According to a third aspect, a method for the joint evaluation of an overall image measured by means of a digital holographic microscope, for example of a biological cell structure, with a first and a second acquisition device in the object space, is proposed, whereby the polarization effect of the biological cell structure is determined by means of a computer device.

[0011] According to a fourth aspect, a method for characterizing a sample is proposed, whereby a sample is measured with a digital holographic microscope.

[0012] Offset refers specifically to a difference in travel time by which wavefronts, particularly of coherent light, are spatially displaced or offset relative to one another. According to this application, an offset set by an optical element, such as a lens, causes a change in the direction of wavefronts relative to each other such that they superimpose at an angle. In English, such an angle is called a shear angle. Offset and angle are physically related, especially via the focal length of the lens causing the change in direction. For example, the relationship "angle is proportional to the quotient of offset / focal length of the lens" can describe this physical relationship.

[0013] The newly proposed solution involves selecting different beam offsets for the different polarization directions of the light field to increase the uniqueness range of interferometric measurements in the interferometer. This offset then results in different fringe widths in the interferogram when the beams are inclined relative to each other. The larger the beam offset is chosen in the interferometer shown in the example, the smaller the fringe width or fringe spacing.

[0014] The advantage of the proposed system lies in the fact that a measurement system with an extended measuring range can be realized with only a few additional components – namely the birefringent plate, the polarizing beam splitter, and the additional camera. Expansion to include multiple wavelengths is possible. Furthermore, the intensity ratio for the images can be adjusted with the different polarization devices by changing the relative polarization of the incident light. If the incident light is not polarized, a polarizer can be added to the beam path for intensity adjustment.

[0015] Due to the asymmetry described above, the advantage here is that a portion of the image in one of the channels also consists of the pure intensity image. The larger the shear angle for this polarization direction, the greater the relative proportion of this intensity image.

[0016] Further advantageous embodiments are claimed in connection with the dependent claims.

[0017] The invention is described in more detail with reference to exemplary embodiments in conjunction with the figures. The figures show: Figure 1, a first embodiment of a Digital Holographic Microscope (DHM); Figure 2, a first embodiment of an offset interferometer; Figure 3, a second embodiment of an offset interferometer; Figure 4, a third embodiment of an offset interferometer; Figure 5, a fourth embodiment of an offset interferometer; Figure 6, a fifth embodiment of an offset interferometer; Figure 7, a sixth embodiment of an offset interferometer; Figure 8, a seventh embodiment of an offset interferometer; Figure 9, an eighth embodiment of an offset interferometer; Figure 10, a ninth embodiment of an offset interferometer; Figure 11, an embodiment of a method according to the invention; Figure 12, an example of a raw image of a stripe pattern; Figure 13, an example of an image of an object without a stripe pattern; Figure 14, an example of a conjugated image;Figure 15 shows an example of a composite image with a first offset; Figure 16 shows an example of the composite image with a second offset; Figure 17 shows an example of the composite image with a third offset.

[0018] The accompanying figures are intended to provide a further understanding of the claimed invention. In conjunction with the description, they serve to explain the concepts and principles of the invention. Other embodiments and many of the mentioned advantages become apparent with reference to the figures. The elements of the figures are not necessarily drawn to scale. Identical, functionally equivalent, and similarly acting elements, features, and components are identified in the figures by the same reference numerals unless otherwise stated.

[0019] Figure 1Figure 1 shows an embodiment of a detection device in the configuration of a digital holographic microscope. It is advantageous to have a small stripe pattern with a high stripe density. In Fourier space—as an embodiment of a conjugate space—this spreads the first orders of the stripe pattern from the zeroth order. The high frequency of the stripe pattern overlaps with the frequency content of the respective object images, which can be, for example, images of cells, such as those shown in Figure 1. Figure 13 The stripe pattern can be extracted from the overall image using further methods. Newer methods utilize a large spread between a first and a zeroth order component in a Fourier image according to... Figure 14, whereby a "synthetic" Fourier space can be enlarged in a cut-out area, resulting in or enabling a high spatial resolution of the reconstructed image.

[0020] Figure 1Figure 1 shows a digital holographic microscope 1 with a first imaging beam path for an interferometer to generate a fringe pattern, and a second imaging beam path for generating a microscopic image, wherein both paths are superimposed and data of a measurement image can be processed in a computer and, in particular, stored in a storage device. On the object side, an objective lens 3 is arranged, which transmits an imaging beam from an object, for example, a cell, via a mirror system 5 and a tube lens 7 as well as lenses L1 and L2 to a camera or detection device 9, wherein 11 denotes a point mirror. Reference numeral 10 denotes an offset mirror. Offset here means a lateral displacement (time difference) by which wavefronts of coherent light are spatially displaced or offset relative to each other.A birefringent plate 13, acting as an optical element for adjusting the offset, is positioned in the second imaging ray path to and from the offset mirror 10. The birefringent plate 13 can be positioned and moved by means of a positioning device 21, in particular rotatable or tiltable. A fringe pattern can be generated. Cropping refers in particular to image processing in which portions of a conjugated image or transformed object image, especially a Fourier image, are cropped out.

[0021] For splitting the light beam in one arm of the interferometer, it is proposed to use a birefringent plate cut from a birefringent crystal with a suitable orientation. The plate can be plane-parallel or wedge-shaped. The splitting depends on the orientation of the optical axes in the birefringent crystal and the thickness of the material. By tilting the plate in the beam, the splitting of the beams can be finely adjusted. With a plane-parallel plate, the beams with the different polarization directions are only laterally displaced. If the plate is wedge-shaped, an angular splitting occurs, depending on the direction of the wedge. This angular splitting further alters the fringe pattern in the interferogram.

[0022] In the interferometer, the split beams are then superimposed with a reference beam. This creates a fringe pattern where the fringe spacing depends on the angle (shear angle) between the measuring beam and the reference wave. In this example, the lateral offset achieved by means of a lens L2 creates an angle between the wavefronts of the generated collimated beams.

[0023] A polarizing beam splitter is then integrated into the beam in front of the camera. The polarization splitting of the beam splitter and the direction of the optical axes in the birefringent crystal should be aligned to each other to obtain optimal results.

[0024] In this setup, there is an asymmetry for the beams: some have parallel polarization vectors due to the wedge angle between the superimposed (collimated) beams, while others have polarization vectors that form a small angle with each other. Because of this small angle, the interferogram then has a small background of non-interfering intensity relative to the fringe pattern of the interferogram.

[0025] If the light source illuminating the optical system in the illustration is unpolarized, its orientation is irrelevant. However, if the light source is polarized or at least partially polarized, the ratio of intensities, and thus how the light is split between the two cameras, can be adjusted by rotating the polarization device of the light source relative to the axes of the birefringent plate or the polarization planes of the beam splitter.

[0026] Each of the images with the different stripe spacing is then captured by a separate camera, each positioned in one of the beam paths behind the beam guide.

[0027] In principle, it is still possible to use more than one wavelength to illuminate the optical system in the image. Everything said above remains valid. The rays from the at least two light sources are then advantageously combined on one axis.

[0028] Since birefringence is also wavelength-dependent, and the fringe spacing for the same optical setup also depends on the wavelength, two additional fringe patterns result with each additional wavelength used – one for each of the polarization directions specified by the beam splitter. This allows the measurement range to be further increased.

[0029] An interferometer is proposed that generates two different fringe patterns via beam separation using polarization. For each pixel, intensity information is thus available for two mutually orthogonal polarization directions. This intensity information can be obtained in two ways: Firstly, directly as a microscope image when the reference beam path is blocked and the interference pattern is suppressed.

[0030] Secondly, from the reconstructed image as an amplitude image after the DHM evaluation.

[0031] Ideally, the images for the two polarization directions should be taken with the same camera orientation, or aligned accordingly after the image has been taken and, if necessary, scaled appropriately to avoid interference effects caused by camera orientation or incorrect image scaling.

[0032] Alternatively, it can be advantageous to align the cameras correctly along the axes, but still shift the pixel patterns by half a pixel diagonal. This allows the lateral resolution to be improved computationally using well-known methods of so-called pixel shift techniques.

[0033] With the chosen DHM setup using a shear interferometer, two shear patterns with different shear angles can be used instead of two wavelengths. These different shear angles result in two interference systems with different periods in depth and lateral separation. This situation is completely comparable to generating a synthetic wavelength using two wavelengths. Here, too, the uniqueness range increases to the least common multiple of the periods of the interference pattern of the two shear interference systems. The freely selectable parameters here are the shear angles of the two interference systems and their difference angles.

[0034] Figure 2 shows a first embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1This digital metadata (DHM) system is of Michelson design. A polarizing beam splitter 17 is arranged in the first imaging beam path after the interferometer. In beam paths downstream of the polarizing beam splitter 17, a first acquisition device 9 simultaneously acquires the first composite image, and a second acquisition device 19 simultaneously acquires a second composite image. The device for generating the at least one further composite image is a birefringent plate 13, which is made primarily of calcite. The birefringent plate 13 is designed here to be plane-parallel, but can alternatively be wedge-shaped.

[0035] Depending on the location of the beam splitting in the beam path, it can be magnified even further at the subsequent optics.

[0036] This is in Figure 2This is clearly visible. When beam splitter plates are placed in a divergent beam, they always have an effect on imaging errors, especially spherical aberration. These effects are not shown in the symbolic beam paths. The beam path is also only shown schematically in the plane. In reality, it may be necessary to orient the polarizing beam splitters at an angle of less than 45 degrees and leave the plane of the drawing, thus requiring the setup to be extended into 3D.

[0037] Figures 2 and 5 show that the proposed modifications are applicable to various types of interferometers. In the interferometer, the light beam is split at a suitable point, and at least one of the two beam segments is laterally displaced. This beam displacement results in different interferograms being formed in the image plane for the two beam segments.

[0038] If the reference wave is generated in the interferometer by a pinhole aperture or a point-shaped mirror, it is advantageous if the lateral offset is generated at one point, so that a single reference mirror can still be used for both interferograms.

[0039] To generate exactly two different interferograms, it is proposed that beam splitting be achieved through birefringence. With a non-polarizing beam splitter, it is no longer possible to completely separate the two partial beams further down the beam path and thus completely decouple the interference patterns. In fact, there is a risk that the at least three waves will interfere with each other in pairs, leading to a superposition of three interference patterns. These three patterns can only be separated with considerable effort.

[0040] In the embodiment according to the claimed invention with polarized beam splitting, a polarizing beam splitter is provided in front of the camera, separating the two interference systems and distributing them between the two different cameras. This makes it possible to record both interference systems simultaneously if the exposure times of the two cameras are synchronized. Alternatively, and thus deviating from the claimed invention, only one camera could be provided, along with a polarization selection element such as a polarization filter or beam splitter, which allows the desired polarization direction to be selected. Electro-optical components for selecting the polarization direction would also be conceivable, or the polarization selection element could be rotated / moved into the desired polarization position.

[0041] With serial image acquisition, it would be just as possible to simply move the adjustment unit for the angle, which can also be called shear, between shots. If the wavelength is known, the shear can be calculated directly from the image containing the interference fringes.

[0042] If the light beams used to generate the interferograms have polarization vectors for linear polarization that are not perpendicular to the plane in which the beams are sheared or oriented at an angle to each other, the image on the camera is not completely modulated. Even if the angles are small, certain small components from each of the individual sheared beams remain in the intensity image and superimpose on the interference pattern on the camera. This effect can be influenced by choosing the geometric design of the setup for the polarization direction and the shear plane, thus determining the contribution of each component to the intensity pattern in the image. The intensity component from the reference beam can be computationally compensated during evaluation, as it is known from the reference image. The camera image then only shows the superposition of the interference pattern and the microscopic image of the object.Adjusting the intensity ratios of the two images is helpful to ensure that sufficient information or signal is available for displaying small structures, such as those found in white blood cells.

[0043] In the following, references to the above reference numerals will refer to the same components.

[0044] Figure 3 shows a second embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1 The device for generating at least one further overall image is here a birefringent material layer 15 on an offset mirror 10.

[0045] Figure 4 A third embodiment of an offset interferometer is shown, in particular as a component of a digital holographic microscope 1 according to

[0046] Figure 1The birefringent plate 13 is positioned here in the second imaging ray path to and from an offset mirror 10.

[0047] Figure 5 A fourth embodiment of an offset interferometer is shown, in particular as a component of a digital holographic microscope 1 according to Figure 1 . An offset interferometer component is created here in a Mach-Zehnder design.

[0048] Figure 6 shows a fifth embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1 . A birefringent plate 13 is arranged here in a beam path after a beam path length symmetrization unit.

[0049] Figure 7 shows a sixth embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1. A birefringent plate 13 is arranged here in a beam path in front of a beam path length symmetrization unit.

[0050] Figure 8 shows a seventh embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1 . A birefringent plate 13 is arranged here in a beam path in front of a beam splitter.

[0051] Figure 9 shows an eighth embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1 . A birefringent plate 13 is arranged here in a beam path after a point mirror.

[0052] Figure 10 shows a ninth embodiment of an offset interferometer, in particular as a component of a digital holographic microscope 1 according to Figure 1. A birefringent plate 13 is arranged here in a beam path in front of a point mirror.

[0053] Figure 11 Figure 1 shows an embodiment of a method according to the invention. The figure shows steps of variable adjustment of an offset, in particular by tilting a birefringent plate, carried out by means of a computer device, wherein in a first step S1 a first offset is generated and in a second step S2 a second offset is generated.

[0054] Figure 12 Figure 1 shows a representation of a raw image of a fringe pattern associated with an interferometer. This image may have been generated using a detection device according to the invention.

[0055] Figure 13Figure 1 shows an image of biological cells as an exemplary embodiment of an object, without a striped pattern. This image may have been generated using a detection device according to the invention. Such an image has a high resolution in the image plane.

[0056] Figure 14 Figure 1 shows an exemplary typical representation of Fourier images of measured composite images of biological cells. This image can be generated from the measured composite image using a computer device according to the invention. The arrow points to a circular diffraction pattern Z of a biological cell. The middle circle represents a zeroth diffraction order 0, and the upper and lower circles represent a symmetrical first diffraction order of +1 and -1, respectively.

[0057] Figure 14Figure 1 shows an embodiment of a conjugated image in the form of a Fourier image, here for example from a measured overall image of a cell structure, wherein the Fourier image has two imaging components: one of a striped pattern with three locations, and one of an object, in this case a cell structure, which is centered at one of the three locations. The invention enables an effective separation of the two imaging components or imaging modes in the Fourier image. The three locations of the striped pattern are symmetrical about the central location for each striped pattern. The pattern of the object depends on its spatial frequency content. Therefore, the radial distance to the origin is relevant for reproducing the internal structure of the object. The pattern of the object is symmetrical only about the central zero point of the zero frequency of the Fourier plane.

[0058] Figure 15shows an example of an overall image with an initial offset.

[0059] Figure 16 the example of the overall picture according to Figure 15 with a second offset.

[0060] Figure 17 the example of the overall picture according to Figure 15 when the overall image is captured simultaneously with two different beam offsets that are superimposed.

[0061] In this Figure 17 A long-wavelength pattern of the synthetic shear or angle is visible, and within it a short-wavelength pattern with lower contrast resulting from the superposition of the two short-wavelength patterns. The long-wavelength structure can also be used for phase unwrapping, with an extended depth range analogous to that achieved with synthetic wavelengths.

[0062] Important preferred control variables in the design include, for example: Coherence length: large enough for the measurement range, but not too large to prevent interference from scattering in the beam path. Shear angle: large enough for sufficiently dense line patterns, but not too large to ensure that the coherence of the light source is sufficient to produce an interference pattern with sufficient contrast across the entire detector.

[0063] Any polarizing beam splitting present adds to the shear splitting. The splitting effect can be partially averaged between the two polarization directions during setup adjustment. Further compensation can be achieved by appropriately selecting the slow and fast axes for splitting. Typically, the slow axis is positioned so that this beam travels along the inner path.

[0064] When illuminating the digital human-mechanical detector (DHM), it is preferable to use either unpolarized lighting or, if fully or partially polarized, lighting that is set at an angle of less than 45 degrees to the axes used for beam splitting. Polarization can also be used to distribute the intensity between the two beam paths, which can further increase the dynamic range of the measurements. For example, the measurement ranges of cameras with non-linear sensitivity—so-called linear / log cameras—can be combined, thus extending the linear range by a factor of two. Alternatively or additionally, the HDR (High Dynamic Range) technique can also be used with the camera.

[0065] By choosing the polarization direction for linearly polarized light sources, or by selecting the preferred polarization direction for partially polarized light sources, the contrast for the object wave components in the microscopic image can also be influenced to some extent. If the optics are not polarization-preserving, the pre-orientation can also depend on the beam path, and such effects should be taken into account when adjusting the system. This effect is only mentioned here in principle because it can have a certain influence on the measurement quality.

[0066] To process the fringe patterns (also known as stripe patterns) with their different fringe periods, the fringe patterns should ideally have the same amplitude or, alternatively, the same modulation. This can be achieved by appropriately scaling the images relative to each other. This scaling can also compensate for any deliberately set intensity differences to obtain the best possible synthetic fringe pattern. It is important to note that the pattern must be of sufficient quality to allow the synthetic fringe pattern to be used for phase unwrapping. For precise phase analysis, the fringe pattern with the most favorable signal-to-noise ratio at the respective measurement location can then be used. This allows for high-resolution measurements despite dynamic range adjustments or expansions.

[0067] If more than two fringe patterns are required that can be detected independently, the beam path before the interferometer can be split, and the split beam directed into a second interferometer with up to two patterns and two cameras. This significantly extends the synthetic measurement range achievable via the fringe patterns. It should be noted that the depth of field of the imaging optics is limited. The second interferometer can then be focused on a different object plane independently of the first interferometer.

[0068] According to the claimed invention, the procedure is such that the first imaging mode produces an image with beam splitting with shear in a first direction and the second imaging mode produces an image with beam splitting with shear in a second direction.

[0069] In this case, the beam splitting for the imaging modes can also be achieved with an amplitude and / or phase grating, for example, a diffraction grating that is not particularly restricted, and / or a birefringent element. According to certain embodiments, a birefringent optical element, e.g., the birefringent plate 13 and / or the birefringent material layer 15, is arranged in the beam direction in front of and / or behind the grating. However, according to certain embodiments, a birefringent optical element, e.g., the birefringent plate 13 and / or the birefringent material layer 15, is used to split the beams (with the shear).

[0070] According to the claimed invention, the first imaging mode produces an image with beam splitting with shear in a first direction, and the second imaging mode produces an image with beam splitting with shear in a second direction, wherein several birefringent optical elements, e.g., 2, 3, 4, or more, are used. These can then be arranged differently, e.g., at an angle of rotation, e.g., 90°, wherein the birefringent optical elements can be adjacent to one another or separated and located at different positions in the beam path. The birefringent optical elements can, for example, be those mentioned in the invention.

[0071] A further advantage of using two interferometers arises when they are arranged with a shear plane orthogonal to each other. This allows the contour of each object point to be imaged with and without shear in at least one direction.

[0072] The approach shown here with the two interferometers can theoretically be extended to n interferometers – possibly with the aforementioned boundary conditions. The separation of the beam path can be achieved by dividing the intensity with a predetermined division ratio, by beam splitting using polarization, or dichroic splitting according to the wavelength of the light, or by combinations thereof.

[0073] The digital holographic microscope according to the claimed invention can be used to measure various samples. Consequently, the present invention also relates to the measurement of a sample with the device according to the invention. In particular, a depth measurement of the sample can also be performed.

[0074] The digital holographic microscope according to the claimed invention enables improved sample acquisition with an enhanced measurement accuracy. This is particularly advantageous for biological and / or medical samples.

[0075] A sample, in this context, is a sample containing an object for which measurement with the device according to the invention is of interest. The sample is not specifically limited and can be provided in a suitable manner, for example, as an in-vitro and / or in vivo sample in the case of medical samples. The sample can, in particular, comprise biological and / or medical samples, which are not specifically limited.

[0076] Biological samples are defined here as samples derived from a living organism, for example, an animal (including humans), plant, fern, fungus, etc., such as cells, cell clusters, cell agglomerates, etc. The origin of the biological sample is not particularly restricted. It is also possible that the biological sample is pretreated before measurement with the device according to the invention, for example, washed, isolated, fixed, etc., for instance, in the preparation of thin-layer or thick-layer samples. Thin-layer techniques can be used, for example, to detect monolayers of cells, while thick-layer techniques can also detect superimposed layers of several cells of the same or different types.

[0077] Medical samples include samples derived from bodily fluids or tissue samples, such as tissue sections, for example, in pathology or histology. Body fluids are not limited to any specific fluid, encompassing liquids, emulsions, and / or suspensions originating from the bodies of living beings, including patients. This includes all fluids excreted by or circulating within the body. Examples of body fluids are blood, urine, saliva, plasma, serum, etc., as well as secretions. The sample may originate from a patient, thus representing a clinical isolate. The sample may come from vertebrates, thus also pertaining to veterinary medicine, or it may be a human sample. Solids, sediments, and / or microcrystals within such bodily fluids and / or secretions can also be measured.

[0078] For the methods according to the invention, it is not excluded that a sample is treated before measurement with the device according to the invention, for example filtered, extracted, washed, diluted, mixed with reagents, etc.

[0079] Various methods are also disclosed with which an overall picture obtained in the measurement method according to the invention is further analyzed with regard to information obtained therein.

[0080] A method for capturing an overall image containing superimposed information components of different imaging modes and measured by means of a digital holographic microscope, in particular of a sample, for example a biological cell structure, a cell agglomerate, a medical sample, and / or of solids, sediments and / or microcrystals in body fluids and / or secretions, in object space, is disclosed, comprising the steps of variably adjusting an offset, in particular by tilting a birefringent plate (13), which are carried out by means of a computer device.

[0081] The computer setup is not particularly limited here, as in other methods according to the invention.

[0082] In this method, the sample is not particularly limited and includes, for example, samples for medical and / or biological applications, such as undiluted body fluids or secretions, which may contain agglomerates of cells as well as accumulations of substances in the form of sediments or solids such as microcrystals, for example in urine, saliva, or bile. An extended depth measurement range is particularly useful for measurements on undiluted fluids or secretions, as it allows for measurement even when cells are at least partially overlapping.

[0083] Depth measurement is also of interest in thin-film and thick-film analyses, for example of blood, as well as in tissue sections. Cells can be present in suspension or dried form on solid or flexible substrates (e.g., glass or plastic). Therefore, in a measurement using the device according to the invention, the sample can also be a thin film, a thick film, or a tissue sample. In this way, cells or tissue components can be identified and differentiated.

[0084] In addition, large cells, cell clusters and / or deposits or solids and microcrystals in body fluids and secretions can also be measured and analyzed in the overall picture according to the invention. Likewise, temporal processes, for example, can be resolved using the methods according to the invention through consecutive measurements, such as blood coagulation processes, e.g., the formation of cell agglomerations from platelets.

[0085] The measurement in the measurement method according to the invention can be carried out with one or with several different wavelengths.

[0086] Extending the measurement range with the same wavelength during measurement offers a significant advantage over a synthetic wavelength derived from different fundamental wavelengths: it minimizes or eliminates potential measurement errors caused by material dispersions. Furthermore, it also minimizes the impact of absorption bands present in the sample (e.g., a fluid or secretion), or those altered or introduced (e.g., via medications in the secretion or fluid), on the measurement.

[0087] Extending the measurement range by one wavelength also has the advantage that a larger proportion of the visible spectrum can be used for absorption, excitation or emission for additional microscopic measurements, such as absorption measurements or fluorescence measurements, which are performed synchronously with an extended digital holographic microscope.

[0088] Also disclosed is a method for the joint evaluation of a composite image, for example of a biological cell structure, measured by a digital holographic microscope, using a first and a second acquisition device in the object space, wherein the polarization effect of the biological cell structure is determined by means of a computer. Here, too, advantages arise from measuring a sample with the device according to the invention when evaluating the composite image. However, the sample with which the composite image is generated is not limited to a biological cell structure, but generally includes samples in which separate imaging with different polarization directions produces an effect.

[0089] In the methods for analyzing the entire image, the measured image can also be digitally focused, and the type of focusing is not particularly limited. For example, image stacks can also be measured for this purpose.

[0090] In this way, for example, one can study the cell nucleus from the level of the dried cells in thin films, or select corresponding measurement levels of interest, for example in cell aggregates and / or crystals, e.g. a doctor during a later evaluation.

Claims

1. Digital holographic microscope (1) having an interferometer for capturing and processing a first overall image of a measurement object that was measured with a first offset, having a device for generating at least one further overall image of the measurement object with at least one offset that differs from the first offset, and having a first imaging beam path for a first imaging mode and a second imaging beam path for a second imaging mode, wherein information portions of the first imaging mode and of the second imaging mode are overlayed in the first and the further overall image of the measurement object; wherein the first imaging mode generates an image with beam splitting with shear in a first direction, and the second imaging mode generates an image with beam splitting with shear in a second direction, wherein a polarizing beam splitter (17) is arranged downstream the interferometer in the first imaging beam path, wherein in the beam paths downstream of the polarizing beam splitter (17), a first capture device (9) captures the first overall image of the measurement object in the object space and, simultaneously, a second capture device (19) captures a second overall image of the measurement object in the object space.

2. Digital holographic microscope (1) according to Claim 1, characterized in that both paths are at least partially overlaid, and data of an overall image can be edited in a computer device and, in particular, can be stored in a memory device.

3. Digital holographic microscope (1) according to Claim 1 or 2, characterized in that beam splitting for the imaging modes is implemented using an amplitude and / or phase grating, and a birefringent optical element is used to split the beams.

4. Digital holographic microscope (1) according to any of Claims 1 to 3, characterized in that the device for generating the at least one further overall image of the measurement object comprises a birefringent optical element for beam splitting with shear.

5. Digital holographic microscope (1) according to Claim 4, characterized in that the birefringent optical element is a birefringent plate (13) comprising calcite, wherein the birefringent plate (13) has a plane-parallel or wedge-shaped embodiment.

6. Digital holographic microscope (1) according to Claim 5, characterized in that the birefringent plate (13) is positioned in the second imaging beam path.

7. Digital holographic microscope (1) according to Claim 6, characterized in that the birefringent plate (13) is positioned in the second imaging beam path to and / or from an offset mirror (10).

8. Digital holographic microscope (1) according to Claim 4, characterized in that the device for generating the at least one further overall image comprises a birefringent material layer (15) on an offset mirror (10), wherein the birefringent material layer (15) has a plane-parallel or wedge-shaped embodiment.

9. Digital holographic microscope (1) according to any of the preceding claims, characterized in that the interferometer is created as a Michelson-type or as a Mach-Zehnder-type construction.

10. Method for capturing, in the object space, an overall image of a measurement object that contains overlaid information portions from different imaging modes and is measured by means of a digital holographic microscope (1) according to any of the preceding claims, the method including the following steps carried out by means of a computer device: setting an offset variably, and capturing the overall image of the measurement object using a capture device.

11. Method for evaluating an overall image of a measurement object that was measured by means of a digital holographic microscope (1) according to any of Claims 1 to 9, wherein the polarization effect of the measurement object is determined by means of a computer device.

12. Method according to Claim 10 or 11, wherein the measured overall image is digitally scanned through the focal planes.

13. Method for characterizing a sample, wherein a sample is measured using a digital holographic microscope according to any of Claims 1 to 9.