WO2019007474A1

WO2019007474A1 - Differential digital holographic microscopy

Info

Publication number: WO2019007474A1
Application number: PCT/EP2017/066422
Authority: WO
Inventors: Thomas Engel
Original assignee: Siemens AG; Siemens Corp
Current assignee: Siemens AG; Siemens Corp
Priority date: 2017-07-03
Filing date: 2017-07-03
Publication date: 2019-01-10
Anticipated expiration: 2020-01-03

Abstract

The present invention pertains to differential digital holographic microscopy (DHM), wherein two birefringent plates (6 and 7) split up a sample light beam (5) emanating from an illuminated sample (3) in two different split directions, preferably orthogonal to each other, to physically create differential interference holograms of the sample. The solution of the present invention improves the imaging capabilities of conventional microscopes employing only a single birefringent crystal with only a single split direction by splitting up a sample light beam in different orientations allowing to image structures arbitrarily oriented in the image plane.

Description

Differential Digital Holographic Microscopy The present invention pertains generally to digital holo^¬ graphic microscopy (DHM) . The present invention relates par^¬ ticularly to DHM for hematology applications and cytological diagnostics. The various systems, methods, and apparatuses described herein may be applied to, for example, red blood cell (RBC) volume measurement and white blood cell (WBC) dif^¬ ferential (cell type classification) applications. However, the present concepts are not limited to these applications and may be applied to general microscopic inspection. In the healthcare sector, hematology is a very prominent field of diagnostics. One important effort in this field is directed at optically imaging red blood cells and/or white blood cells and resolving them in a three dimensional way in order to analyze and/or distinguish the inner cell structure. To this end, there is a need to analyze cell samples in a non-destructive and non-detrimental way. The ability of digi^¬ tal holographic microscopy (DHM) to achieve high-resolution, wide field imaging with extended depth and morphological in^¬ formation on living cells in a potentially label-free manner without the need for markers or dyes positions the technology for use in several clinical applications. The possibilities of DHM have increased during the last years due to an in^¬ crease in the development of digital sensors and computers. Unlike conventional microscopes, such as phase contrast mi- croscopes or confocal microscopes, both phase contrast and amplitude contrast information may be recorded simultaneous^¬ ly. Thus, for example, in the area of hematology, DHM may be used for RBC volume measurement, WBC differential applica^¬ tions and similar use cases. Common path interferometers have proven to be very well suit^¬ ed for DHM as they are very robust despite an interferometric image being involved. Typically, there are basically two classes of interferometers utilized, absolute interferometers as, for example, taught by WO 2013/140396 Al or WO

2017/034517 Al, and differential setups as, for example, taught by WO 2013/011000 Al or EP 1631788 Bl . However, there is a need to find a measuring setup with high imaging performance and low complexity paired with usability and stability, that is, a system with high efficiency.

Oh, Chulwoo et al . , "On-Chip Differential Interference Con- trast Microscopy Using Lensless Digital Holography, " Optics Express, 18(5): 4717-4726, 2010, suggest the use of a

birefringent crystal with lensless digital holography to cre^¬ ate an on-chip differential interference contrast (DIC) mi^¬ croscope. The key element to this idea is to use a thin birefringent crystal to split up a light beam into two paral^¬ lel light beams with orthogonally polarized field components and subsequently physically create differential interference holograms at a sensor plane that encode the spatial phase variation of a sample into amplitude oscillations. Using an incoherent source with a large aperture, in-line holograms of micro-objects are created, which interact with a uniaxial crystal and an absorbing polarizer, encoding differential interference contrast information of the objects on a chip. In this context microscope means single digit micrometer resolu- tion as related to the pixel size of the detector employed.

Despite the fact that an incoherent source with a large aper^¬ ture is used, holographic digital processing of such holo^¬ grams rapidly recovers the differential phase contrast image of the specimen over a large field-of-view . Against this background, it is one objective of the present invention to find solutions for DHM with improved imaging ca^¬ pabilities .

This objective is achieved by differential digital holograph^¬ ic microscope having the features of claim 1 and a method for differential digital holographic microscopy having the fea^¬ tures of claim 15.

According to a first aspect of the invention, a differential digital holographic microscope is provided. The microscope comprises a light source configured to illuminate a sample. The microscope further comprises a collecting lens arrange- ment configured to collect and focus a sample light beam ema^¬ nating from the illuminated sample. The microscope further comprises a first birefringent plate configured to split up the focused sample light beam by double refraction into a first ordinary beam and a first extraordinary beam in a first split direction. The microscope further comprises a second birefringent plate configured to split up the first ordinary beam and/or the first extraordinary beam, or the focused sample light beam by double refraction into second ordinary beams and second extraordinary beams in a second split direc- tion, wherein the second split direction is different from the first split direction. The microscope further comprises an imaging lens arrangement configured to focus the second ordinary beams and the second extraordinary beams, and, in case the focused sample light beam is split up by the second birefringent plate, the first ordinary beam and the first ex^¬ traordinary beam. The microscope further comprises at least one digital recording device configured to record the focused second ordinary beams and/or second extraordinary beams, and, in case the focused sample light beam is split up by the se- cond birefringent plate, the first ordinary beam and the first extraordinary beam, as a differential interference hol^¬ ogram of the sample. According to a second aspect of the invention, a method for differential digital holographic microscopy is provided. The method comprises illuminating a sample with a light source. The method further comprises collecting and focusing a sample light beam emanating from the illuminated sample with a col- lecting lens arrangement. The method further comprises split^¬ ting up the focused sample light beam by double refraction with a first birefringent plate into a first ordinary beam and a first extraordinary beam in a first split direction. The method further comprises splitting up the first ordinary beam and/or the first extraordinary beam, or the focused sample light beam, by double refraction with a second

birefringent plate into second ordinary beams and second ex^¬ traordinary beams in a second split direction different from the first split direction. The method further comprises fo- cusing the second ordinary beams and the second extraordinary beams, and, in case the focused sample light beam is split up by the second birefringent plate, the first ordinary beam and the first extraordinary beam, with an imaging lens arrangement. The method further comprises recording the focused se- cond ordinary beams and the second extraordinary beams, and, in case the focused sample light beam is split up by the se^¬ cond birefringent plate, the first ordinary beam and the first extraordinary beam, with at least one digital recording device as a differential interference hologram of the sample.

One idea of the present invention is to improve the imaging capabilities of known microscopic solutions, which use at best only one single birefringent crystal with only one split direction, by providing two birefringent plates that permit to split up a light ray in different orientations allowing to image structures arbitrarily oriented in the image plane. For example, the DIC method mentioned above uses white light il^¬ lumination with one single birefringent crystal thus provid- ing one single split direction. For reflected light DIC mi^¬ croscopes, the birefringent plate gets penetrated by the light twice and also in opposite directions. Therefore the illuminating beam is split up and laterally shifted by the first birefringent plate and then interacts with the sample. The part of the light which gets reflected back into the op^¬ tics penetrates the birefringent plate in opposite direction and thus the beam split gets compensated. Further, the phase information of the beam at adjacent sample locations gets transferred into amplitude or intensity modulation at least after the application of a polarization filter device. In transmitted light DIC microscopes, there are separate optical sub-systems for illumination and imaging. Accordingly, there are two birefringent plates, one in the illumination subsys^¬ tem and one in the imaging subsystem. These two plates are aligned with respect to each other to split up the beam in front of the sample and to recombine the beam properly after the sample. Different magnifications for the imaging and il^¬ lumination subsystems can influence the design and size of the birefringent plates. In contrast, the inventive idea of the systems, methods, and apparatuses described herein is to utilize two independent birefringent plates to provide two different split directions with the same or different magni^¬ tudes. One birefringent plate splits up a light beam or light ray only in one direction into two orthogonally polarized beams. The present teaching now provides two independent and different split directions, e.g. rotated with respect to each other by 90°. For example, consider a sample with first line^¬ ar structures oriented orthogonal to a split direction and second linear structures oriented parallel to the split di- rection. Due to the split up of the respective birefringent plate, interferences may be generated between the wavefront of the ordinary beam and the wavefront of the extraordinary beam, the latter being laterally shifted by a small quantity. The information contained by the interferogram relates to the wavefront derivative in this lateral shift direction. Thus, the first linear structures oriented orthogonal to the split direction will feature in the interference pattern, while the second linear structures oriented parallel to the split di- rection will not experience any lateral shift and thus will not be visible in the microscopic image. The present inven^¬ tion now eliminates this drawback by splitting up the beam in two independent and different directions, thereby generally improving the imaging capabilities. The setup according to the invention may be provided in a very compact way that may be manufactured very easily and cost-effectively. The micro^¬ scope of the present invention features a high depth of field compared to standard DIC microscopes due to a comparably low aperture as well as low to zero numerical aperture illumina- tion.

The birefringent beam separation itself is dependent on the material of the birefringent plate, on the thickness of the birefringent plate, on the orientation of the optic axis of the birefringent plate, i.e. the optic axis of the crystal, and on the angle of incidence of the sample light beam. on-, low- and highly coherent light sources may be used with the present teachings. The person of skill will be readily aware that further modifications are possible. For example, the birefringent beam separation may be overlaid with the dif^¬ fraction of a non-zero angle of incidence, that is, the sam^¬ ple light beam may have various angles of incidence on the birefringent plates. Advantageous embodiments and improvements of the present in^¬ vention are found in the subordinate claims.

According to an embodiment of the invention, the second split direction may be rotated by a rotation angle with respect to the first split direction. The rotation angle may be equal to 90° .

According to an embodiment of the invention, the second birefringent plate may be arranged adjacent to the first birefringent plate.

According to an alternative embodiment of the invention, a beam splitter may be arranged between the sample and the first birefringent plate. The beam splitter may be configured to split the sample light beam onto the second birefringent plate. Thus, in contrast to an embodiment where the second birefringent plate is arranged adjacent to the first

birefringent plate, the two birefringent plates may be ar- ranged apart and independently of each other.

According to an embodiment of the invention, the microscope may further comprise a polarizer arranged between the imaging lens arrangement and the at least one digital recording de- vice. The polarizer may be configured to project the polari^¬ zation of at least one of the second ordinary beams and at least one of the second extraordinary beams on a common axis. The common axis may be oriented under an angle of 45° with respect to the polarization of the respective second ordinary beams and respective second extraordinary beams. Since polar^¬ ization is used in the birefringent plates, the two beams split up can no longer interfere and thus two images are overlaid incoherently. The two images are displaced according to the thickness of the birefringent plate material, the an- gle of incidence and the type of birefringent material. In this embodiment, a polarizer is introduced to get a common projection from the two orthogonally polarized and split up beams on a common axis, which may be for example oriented un- der 45 degrees with respect to the orientation of the polari^¬ zation axis of the two orthogonally polarized beams. A dif^¬ ferent orientation from 45 degrees may be chosen however, e.g. if one of the two polarized images should have a higher intensity. This will create an offset for one of the micro- scopic images, which might be beneficial if the image content is later used to display a microscopic image on the one hand and to calculate the differential phase from the overlay of the two images on the other hand. The polarizer thus may en^¬ hance the image contrast in a projected polarization direc- tion.

According to an embodiment of the invention, the microscope may further comprise a retardation plate arranged between the imaging lens arrangement and the at least one digital record- ing device. The retardation plate may be configured to rotate the polarization of at least one of the second ordinary beams and at least one of the second extraordinary beams such that the respective second ordinary beams and respective second extraordinary beams at least partially interfere with each other. Thus, the retardation plate is introduced in order to rotate the plane of polarization of the two beams and thus make the beams at least partially interfere with each other. In case the effect of the retardation plate may be too weak, because the wavelength of the two polarized beams and their respective bandwidth is identical, a multiple order retarda^¬ tion plate may be provided instead of a low or a single order retardation plate. According to an embodiment of the invention, the microscope may further comprise an imaging beam splitter arranged between the imaging lens arrangement and the at least one digi^¬ tal recording device. In order to enhance image contrast or to avoid a retardation plate, a polarizing beam splitter may be used. The axis of the polarizing beam splitter may for ex^¬ ample be oriented under 45 degrees with respect to the polar^¬ ization direction as defined by the birefringent plate. In this case one gets two projections from each of the two po- larized and split up beams that are superimposed. Alterna^¬ tively, a polarizer can be uses in combination with a normal beam splitter.

The imaging beam splitter may be configured as a polarizing beam splitter. The imaging beam splitter may be configured to project the polarization of at least one of the second ordi^¬ nary beams and at least one of the second extraordinary beams on a common axis. The common axis may be oriented under an angle of 45° with respect to the polarization of the respec- tive second ordinary beams and respective second extraordi^¬ nary beams .

According to an embodiment of the invention, at least two digital recording devices may be provided. Each digital re- cording device may be configured to record a second ordinary beam and a second extraordinary beam. The digital recording device (s) may be, for example, digital cameras or digital sensors or the like. According to an embodiment of the invention, the microscope may further comprise a monitoring beam splitter arranged between the sample and the first birefringent plate. The moni^¬ toring beam splitter may be configured to split a monitoring light beam from the sample light beam. The microscope may further comprise a monitoring lens arrangement configured to focus the monitoring light beam. The microscope may further comprise a monitoring digital recording device configured to record the focused monitoring light beam as a microscopic im- age of the sample. In this advantageous embodiment, a conven^¬ tional microscopic image of the sample is provided in addi^¬ tion to the differential digital hologram. This additional microscopic image may be used to monitor the sample while re^¬ cording the holographic image of the sample. Thus, a hybrid setup of conventional microscope and differential digital holographic microscope is provided.

According to an embodiment of the invention, at least one of the first birefringent plate and the second birefringent plate may be formed from at least one of calcit and quartz or other suited materials known to the person of skill.

The invention will be explained in greater detail with reference to exemplary embodiments depicted in the drawings as ap- pended.

The accompanying drawings are included to provide a further understanding of the present invention and are incorporated in and constitute a part of this specification. The drawings illustrate the embodiments of the present invention and to^¬ gether with the description serve to explain the principles of the invention. Other embodiments of the present invention and many of the intended advantages of the present invention will be readily appreciated as they become better understood by reference to the following detailed description. The ele^¬ ments of the drawings are not necessarily to scale relative to each other. In the figures, like reference numerals denote like or functionally like components, unless indicated other^¬ wise . Fig. 1 schematically shows a differential digital holographic microscope according to an embodiment of the invention. Fig. 2 shows a schematic flow diagram of a method for differential digital holographic microscopy according an embodiment of the invention.

Fig. 3 schematically shows a differential digital holographic microscope according to another embodiment of the invention.

Fig. 4 schematically shows a differential digital holographic microscope according to yet another embodiment of the inven- tion .

Fig. 5 schematically shows a differential digital holographic microscope according to yet another embodiment of the inven- tion . Fig. 6 schematically shows a differential digital holographic microscope according to yet another embodiment of the inven- tion .

Fig. 7 schematically shows a differential digital holographic microscope according to yet another embodiment of the inven- tion .

Although specific embodiments are illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a variety of alternate and/or equivalent imple^¬ mentations may be substituted for the specific embodiments shown and described without departing from the scope of the present invention. Generally, this application is intended to cover any adaptations or variations of the specific embodi^¬ ments discussed herein.

Figure 1 schematically shows a differential digital holo- graphic microscope 1 according to an embodiment of the inven^¬ tion, while Fig. 2 shows a schematic flow diagram of a method M for differential digital holographic microscopy according an embodiment of the invention that may be performed, for ex^¬ ample, on the differential digital holographic microscope 1 of Fig.l. The further Figs. 3-7 show various other embodi^¬ ments of the invention.

Fig. 1 shows a basic embodiment of a digital holographic mi^¬ croscope according to the invention. Digital holographic mi- croscopy (DHM) is an imaging technology that provides the ability to quantitatively track sub-nanometric optical thick^¬ ness changes in transparent specimens. Unlike traditional digital microscopy, in which only intensity (amplitude) in^¬ formation about a specimen is captured, DHM captures both phase and intensity. The phase information, captured as a hologram, can be used to reconstruct extended morphological information (such as depth and surface characteristics) about the specimen using a computer algorithm. Modern DHM implementations offer several additional benefits, such as fast scan- ning/data acquisition speed, low noise, high resolution and the potential for label-free sample acquisition.

The shown microscope 1 may be employed for hematology appli^¬ cations and cytological diagnostics and other clinical appli- cations. For example, the microscope 1 may be used for red blood cell (RBC) volume measurement or white blood cell (WBC) differential (cell type classification) applications. Howev^¬ er, the application of this microscope 1 is not limited to these use cases. Various other (clinical) applications may be envisaged including but not limited to sediment analysis, tissue pathology applications, rare cell detection applica^¬ tions and so on. The microscope 1 features a light source 2, which is config^¬ ured to illuminate a sample 3. A sample light beam 5 leaving the illuminated sample 3 is collected and focused by a col^¬ lecting lens arrangement 4. To this end, the collecting lens arrangement 4 may comprise lenses and other optical compo- nents known to the person of skill, e.g. an objective lens, a tube lens etc. A first birefringent plate 6, which may be formed, for example, from quarz or calcit or the like, is placed in the sample light beam 5 so as to split up the fo^¬ cused sample light beam 5 by double refraction into a first ordinary beam 8a and a first extraordinary beam 8b in a first split direction. The ordinary beam 8a stems from light whose polarization is perpendicular to the optic axis of the mate^¬ rial of the first birefringence plate 6. The extraordinary beam 8b on the other stems from light whose polarization is in the direction of the optic axis. A second birefringent plate 7 is placed adjacent to the first birefringent plate 6 in such a way that a second split direction of the second birefringent plate 7 is rotated by 90° with respect to the first split direction of the first birefringence plate 6. The second birefringent plate 7 is thus configured to split up the first ordinary beam 8a by double refraction into a second ordinary beam 9a and a second extraordinary beam 9b. Correspondingly, the second ordinary beam 8b is also split up into a second ordinary beam 9a and a second extraordinary beam 9b. An imaging lens arrangement 10 or general imaging optics is provided to focus the second ordinary beams 9a and the second extraordinary beams 9b, which are then recorded by a digital recording device 11, like for example a digital camera or a sensor chip or the like which can also be sensitive to the polarization of the beams, as a differential interference hologram of the sample 3. The digitized differential inter^¬ ference hologram may consecutively be processed by a micro^¬ processor, a computer or similar (not shown in the figures) in the usual way.

The method M shown in Fig. 2 correspondingly comprises under Ml illuminating the sample 3 with the light source 2. The method M further comprises under M2 collecting and focusing the sample light beam 5 emanating from the illuminated sample 3 with a collecting lens arrangement 4. The method M further comprises under M3 splitting up the focused sample light beam 5 by double refraction with the first birefringent plate 6 into the first ordinary beam 8a and the first extraordinary beam 8b in a first split direction, as well as under M4 splitting up the first ordinary beam 8a and the first ex^¬ traordinary beam 8b by double refraction with a second birefringent plate 7 into the second ordinary beams 9a and the second extraordinary beams 9b in a second split direction different from the first split direction. Further, the method M comprises under M5 focusing the second ordinary beams 9a and the second extraordinary beams 9b with an imaging lens arrangement 10. Finally, the method M comprises under M6 re^¬ cording the focused second ordinary beams 9a and the second extraordinary beams 9b with the digital recording device 11 as a differential interference hologram of the sample 3.

In this way, the imaging capabilities of conventional micro^¬ scopes employing only a single birefringent crystal may be improved by splitting up a sample light beam in different orientations allowing to image structures arbitrarily orient^¬ ed in the image plane. Referring now to the embodiment shown in Fig. 3, a polarizer 13 is introduced to get a common projection from the split up second ordinary beams 9a and the second extraordinary beams 9b on a common axis, which may be for example oriented under 45 degrees with respect to the orientation of the polariza^¬ tion axis of the orthogonally polarized beams. A different orientation from 45 degrees may be chosen as well, however. Thus, the second ordinary beams 9a and the second extraordi^¬ nary beams 9b can be brought to overlap and at least partly interfere with the polarizer such that an overlaid image may be produced. The mixing ratio of the components from the two polarization directions and thus the contrast of the produced image can be adjusted via the direction of the polarization filter axes of the at least partially polarizing filter ele- ment . In a similar vein, an at least partly polarizing beam splitter or the like may be utilized for the same purpose. Alternatively or in addition, instead of the rotation of the filter axes in the beam direction of the polarizer 13, the retardation plate may be rotated around the optical beam axis to adjust the mixing ratio of the polarization components.

Another option would be to use a polarization sensitive digi^¬ tal recording device, e.g. a digital camera, as already dis^¬ cussed with reference to Figs. 1-3. In general, the digitized image may be further processed by computer algorithms.

Figs. 4-6 show alternative embodiments of the invention, in which a beam splitter 12 is placed within the collecting lens arrangement 4, i.e. between the sample 3 and the first birefringent plate 6, such that the sample light beam 5 is split up into two beams, one lead onto the first birefringent plate 6 and the other onto the second birefringent plate 7, each beam being split up into respective ordinary beams and extraordinary beams before being focused and recorded by a digital recording device 11. Also in these embodiments, the split direction of the second birefringent plate 7 is rotated with respect to the split direction of the first birefringent plate 6. Correspondingly, more than one digital recording device 11 (e.g. digital cameras) are employed in these embodi- ments. It will be clear to the person of skill that various variants of the basic embodiments of the invention are possi^¬ ble. Solely for illustration purposes, Figs. 4-6 demonstrate slight variations of the basic setup, wherein, for example, a retardation plate and/or a polarizer can be placed between the sample 3 and the light source 2. The present invention may be generally used with non-, low- and/or highly coherent light sources. Furthermore, it is possible to utilize polar^¬ izers 13, retardation plates 14 and/or beam splitters 12, e.g. a polarizing beam splitter, in the arrangement, in par- ticular for further manipulating the imaging process between the retardation plates 6, 7 and the digital recording de^¬ vice (s) 11. For example, a retardation plate 14 may be ar^¬ ranged between the imaging lens arrangement 10 and the at least one digital recording device 11 to rotate the polariza- tion of at least one of the second ordinary beams 9a and at least one of the second extraordinary beams 9b such that the respective second ordinary beams 9a and respective second ex^¬ traordinary beams 9b at least partially interfere with each other .

Fig. 7 finally depicts a further embodiment of the invention, wherein a monitoring beam splitter 16 is arranged between the sample 3 and the first birefringent plate 6 to split a moni^¬ toring light beam 19 from the sample light beam 5, which then may serve to monitor the sample directly during the imaging process. To this end, a monitoring lens arrangement 17 focus^¬ es the monitoring light beam 19 onto a monitoring digital re^¬ cording device 18, e.g. another digital camera or a monitor or similar, which then records the focused monitoring light beam 19 as a microscopic image of the sample 3, that is, an image that directly depicts the sample and does not neces^¬ sarily require any further digital processing or the like. In the foregoing detailed description, various features are grouped together in one or more examples or examples with the purpose of streamlining the disclosure. It is to be under^¬ stood that the above description is intended to be illustra^¬ tive, and not restrictive. It is intended to cover all alter- natives, modifications and equivalents. Many other examples will be apparent to one skilled in the art upon reviewing the above specification.

The embodiments were chosen and described in order to best explain the principles of the invention and its practical ap^¬ plications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with vari^¬ ous modifications as are suited to the particular use contem^¬ plated. Many other examples will be apparent to one skilled in the art upon reviewing the above specification.

Claims

Patent claims

1. A differential digital holographic microscope (1) compris^¬ ing :

a light source

(2) configured to illuminate a sample

(3) ;

a collecting lens arrangement

(4) configured to collect and focus a sample light beam (5) emanating from the il^¬ luminated sample (3) ;

a first birefringent plate (6) configured to split up the focused sample light beam (5) by double refraction into a first ordinary beam (8a) and a first extraordi^¬ nary beam (8b) in a first split direction;

a second birefringent plate (7) configured to split up the first ordinary beam (8a) and/or the first extraordi^¬ nary beam (8b), or the focused sample light beam (5), by double refraction into second ordinary beams (9a) and second extraordinary beams (9b) in a second split direc^¬ tion, wherein the second split direction is different from the first split direction;

an imaging lens arrangement (10) configured to focus the second ordinary beams (9a) and/or the second extraordi^¬ nary beams (9b), and, in case the focused sample light beam (5) is split up by the second birefringent plate (7), the first ordinary beam (8a) and the first extraor^¬ dinary beam (8b); and

at least one digital recording device (11) configured to record the focused second ordinary beams (9a) and/or se^¬ cond extraordinary beams (9b), and, in case the focused sample light beam

(5) is split up by the second

birefringent plate (7), the first ordinary beam (8a) and the first extraordinary beam (8b), as a differential in^¬ terference hologram of the sample (3) . Microscope (1) according to claim 1, wherein the second split direction is rotated by a rotation angle with re^¬ spect to the first split direction.

Microscope (1) according to claim 2, wherein the rota^¬ tion angle is equal to 90°.

Microscope (1) according to one of the preceding claims, wherein the second birefringent plate (7) is arranged adjacent to the first birefringent plate (6) .

Microscope (1) according to one of the claims 1 to 3, further comprising:

a beam splitter (12) arranged between the sample (3) and the first birefringent plate

(6), wherein the beam splitter (12) is configured to split the sample light beam (5) onto the second birefringent plate

(7) .

Microscope (1) according to one of the preceding claims, further comprising:

a polarizer (13) arranged between the imaging lens arrangement (10) and the at least one digital recording device (11), wherein the polarizer (13) is configured to project the polarization of at least one of the second ordinary beams (9a) and at least one of the second ex^¬ traordinary beams (9b) on a common axis.

Microscope (1) according to claim 6, wherein the common axis is oriented under an angle of 45° with respect to the polarization of the respective second ordinary beams (9a) and respective second extraordinary beams (9b) .

8. Microscope (1) according to one of the preceding claims, further comprising: a retardation plate (14) arranged between the imaging lens arrangement (10) and the at least one digital re^¬ cording device (11), wherein the retardation plate (14) is configured to rotate the polarization of at least one of the second ordinary beams (9a) and at least one of the second extraordinary beams (9b) such that the re^¬ spective second ordinary beams (9a) and respective se^¬ cond extraordinary beams (9b) at least partially inter^¬ fere with each other.

Microscope (1) according to one of the preceding claims, further comprising:

an imaging beam splitter (15) arranged between the imaging lens arrangement (10) and the at least one digital recording device (11) .

Microscope (1) according to claim 9, wherein the imaging beam splitter (15) is configured as a polarizing beam splitter, wherein the imaging beam splitter is configured (15) to project the polarization of at least one of the second ordinary beams (9a) and at least one of the second extraordinary beams (9b) on a common axis.

Microscope (1) according to claim 10, wherein the common axis is oriented under an angle of 45° with respect to the polarization of the respective second ordinary beams (9a) and respective second extraordinary beams (9b) .

Microscope (1) according to one of the preceding claims, wherein at least two digital recording devices (11) are provided, each digital recording device (11) being con^¬ figured to record a second ordinary beam (9a) and a se^¬ cond extraordinary beam (9b) . Microscope (1) according to one of the preceding claims, further comprising:

a monitoring beam splitter (16) arranged between the sample (3) and the first birefringent plate (6), wherein the monitoring beam splitter (16) is configured to split a monitoring light beam (19) from the sample light beam (5) ;

a monitoring lens arrangement (17) configured to focus the monitoring light beam (19); and

a monitoring digital recording device (18) configured to record the focused monitoring light beam (19) as a mi^¬ croscopic image of the sample (3) .

Microscope (1) according to one of the preceding claims, wherein at least one of the first birefringent plate (6) and the second birefringent plate (7) is formed from at least one of calcit and quartz.

A method (M) for differential digital holographic mi^¬ croscopy comprising:

illuminating (Ml) a sample (3) with a light source (2); collecting and focusing (M2) a sample light beam (5) emanating from the illuminated sample (3) with a collect^¬ ing lens arrangement (4);

splitting up (M3) the focused sample light beam (5) by double refraction with a first birefringent plate (6) into a first ordinary beam (8a) and a first extraordi^¬ nary beam (8b) in a first split direction;

splitting up (M4) the first ordinary beam (8a) and the first extraordinary beam (8b), or the focused sample light beam (5) , by double refraction with a second birefringent plate (7) into second ordinary beams (9a) and second extraordinary beams (9b) in a second split direction different from the first split direction; focusing (M5) the second ordinary beams (9a) and the se^¬ cond extraordinary beams (9b), and, in case the focused sample light beam (5) is split up by the second

birefringent plate (7), the first ordinary beam (8a) and the first extraordinary beam (8b), with an imaging lens arrangement (10); and

recording (M6) the focused second ordinary beams (9a) and the second extraordinary beams (9b), and, in case the focused sample light beam (5) is split up by the second birefringent plate (7), the first ordinary beam (8a) and the first ex^¬ traordinary beam (8b), with at least one digital recording device (11) as a differential interference hologram of the sample (3) .