OBJECTS

Technical field

The present invention generally relates to a method, devices, and use of devices for analyzing a sample comprising at least one translucent biological object by means of digital holographic microscopy.

Background of the invention

There exists a never ending demand for additional and more accurate information about biological objects, such as cells, in humans, animals, plants and other organisms. Cells have for a long time been studied by means of light microscopy, such as fluorescence, confocal and phase contrast microscopy. However, when using fluorescence or confocal microscope the cells must be marked or stained and the used marker or stain has a potential toxic effect on the cells that may be influenced and thereby the development of the cells is disturbed. In addition, the marker may be bleached over time, which renders the study of cell development over substantial time ranges difficult. When using fluorescence microscope also the focal plane must be set mechanically and this may be distorted over time by e.g. temperature changes in the ambient environment.

In many cases it is important to be able to study living cells without the risk of influencing the cell and the cell development by toxic markers or stains. Phase contrast microscopy enables the study of living cells without the need for markers. However, phase contrast microscopy does not allow quantification of the phase shift of the studied object, which implies that it is difficult to quantify the area of distribution or the thickness of the cell. The

disadvantages of mechanically setting of the focal plane also apply to the phase contrast microscope. In addition, since the studied object normally has an irregular upper surface the focal plane differs from one spot to another. Thereby, it is not possible to achieve a sharp image of all portions of the object in one image. Several images with varying setting of the focal plane must be produced in order to achieve sharp visualization of all portions of the object. Especially if the studied object has protrusions, the sharpness of one image will be unsatisfactory, since the protrusions will not be in the same focal plane. Digital holographic microscopy enables studies of living cells without the need for markers or stains and enables quantification of the studied objects. The possibilities of digital holographic microscopy have increased during the last years due to the dramatic development of digital sensors and computers.

WO 2009/154558 discloses one way of studying cells by means of digital holographic microscopy. Specifically, WO 2009/154558 discloses an observation vessel which may be used in connection to a method for digital holographic microscopy. The observation vessel comprises first and second holding means which are provided to keep a sample and to be in contact with the sample. The first holding means acts as a lid of the observation vessel and reduces the scattering of light in the upper surface of the sample.

According to WO 2009/154558, the noise in the hologram resulting from the method is thereby reduced.

Although WO 2009/15455 discloses some measures for reducing noise in digital holographic microscopy, there is still a need for an improved technique that will result in holograms with a high quality and accurate information about the studied objects. Summary of the invention

In view of the above, an objective of the invention is to provide accurate phase and amplitude information about translucent biological objects. This accurate information may be utilized to produce high-resolution holographic images of the translucent biological objects.

According to a first aspect, a method for analyzing a sample

comprising at least one translucent biological object by means of digital holographic microscopy is provided. The method comprises

arranging the sample on a first reflecting surface of a sample holder, the first reflecting surface being essentially flat;

creating at least one beam of coherent light, the at least one beam comprising at least one object beam;

directing the at least one object beam towards the first reflecting surface at an angle which is essentially perpendicular to the first reflecting surface, such that the at least one object beam passes through the sample in a first direction, is reflected by the reflecting surface, and passes through the sample in a second direction which is essentially opposed to the first direction; detecting an interference pattern, called hologram, originating from the at least one object beam; and

reconstructing phase and/or amplitude information from the

interference pattern.

Each time the at least one object beam passes through the sample comprising at least one biological object, it will be phase shifted as it experiences a longer, or shorter, optical path. This is due to differences in refractive index between the sample and the surrounding medium which may have a higher or lower refractive index and which thereby causes the object beam to travel a longer or shorter optical path. With the above arrangement, the at least one object beam passes twice through the sample comprising at least one biological object and, consequently, the phase shift becomes twice the phase shift of a beam that only passes the sample once. This is advantageous when detecting the hologram since a larger phase shift is easier to detect and to separate from noise which is also present in the detected signal. In other words, the above method improves the signal-to- noise ratio in the detected signal. Accordingly, the hologram and the phase and/or amplitude information reconstructed therefrom becomes more accurate with the above method.

It is further noted that the above method enables visualization of biological objects without having to mark or stain the objects, which is time consuming and which may affect the objects and thereby the results. Due to this reason, the above method may advantageously be used to study living cells and the development of cells, such as cell growth.

The method may further comprise dividing the at least one beam into the at least one object beam and at least one reference beam; and

superposing the at least one object beam that has passed through the sample with the at least one reference beam, thereby creating an interference pattern. By creating at least one reference beam in this way and by

superposing it with the at least one object beam, an easy way of producing an interference pattern indicative of phase and/or amplitude information is obtained. Further, the reconstruction of phase and/or amplitude information may be made simpler and more efficient if the interference pattern is obtained from at least one object beam and at least one reference beam. Still further, more accurate information concerning the positioning of the at least one translucent object may be obtained in this way. Note that the at least one object beam and the at least one reference beam will be mutually coherent. The method may further comprise arranging the sample in the sample holder between a first holding means having a first inner surface and a first outer surface and a second holding means comprising the first reflecting surface, wherein the first inner surface and the first reflecting surface keep the sample and are in contact with the sample, such that the at least one object beam passes through the first inner and outer surfaces before passing the sample in the first direction and after passing the sample in the second direction. In this way, the sample do not have any free surface in which the incident at least one object beam may be scattered. Thereby, the noise in the detected signal is further reduced.

The method may further comprise directing the at least one reference beam towards a second reflecting surface at an angle which is essentially perpendicular to the second reflecting surface. Thereby, the at least one reference beam may be reflected in the second reflecting surface and directed back towards the at least one object beam in order to be superposed with the at least one object beam.

The steps of dividing, directing the at least one object beam and the at least one reference beam towards the first and second reflecting surfaces, and the superposing may be performed by a beam splitter. Thereby, an easy and flexible way of performing these steps is obtained.

The first outer surface of the first holding means may be semi- translucent, and the method may further comprise arranging the first holding means at an angle relatively the first reflecting surface, such that the first outer surface partly transmits the at least one beam, thereby forming the at least one object beam, and partly reflects the at least one beam, thereby forming the at least one reference beam. With this arrangement, the first outer surface acts as a beam dividing means which divides the at least one beam into the at least one object beam and the at least one reference beam.

Thereby, no extra components are required to divide the at least one beam and hence an easy way of dividing the at least one beam is obtained.

According to a second aspect of the invention, there is provided a digital holographic microscopy apparatus for analyzing a sample comprising at least one translucent biological object. The apparatus comprises

at least one light source arranged to create at least one beam of coherent light, the at least one beam comprising at least one object beam; a sensor arranged to detect an interference pattern;

a beam directing means arranged to direct the at least one beam; and a sample holder adapted to hold the sample, wherein the sample holder comprises a first reflecting surface which is essentially flat and is arranged to reflect the at least one object beam, and wherein the sample, when the apparatus is in use, is arranged on the first reflecting surface;

wherein the light source, the beam directing means and the sample holder are arranged relatively each other such that, when in use, the at least one object beam is directed towards the first reflecting surface at an angle which is essentially perpendicular to the first reflecting surface, and such that the at least one object beam that has passed through the sample is directed towards the sensor, thereby creating an interference pattern at the sensor.

The features and advantages of the first aspect generally applies to the second aspect. Moreover, it is noted that no expensive microscope lenses are required in the above apparatus in contrast to other types of microscopes. Rather, the only optical components required, such as the sensor and the at least one light source, are cheap standard components. As a result, the apparatus is cheap to produce. Further, as the number of optical components are minimized, the number of noise sources are reduced, thereby resulting in more accurate measurements.

Different setups may be used in digital holographic microscopy. For example, a so-called in-line digital holography setup may be used in which the interference pattern originates from a single beam of light, such as the at least one object beam. If an in-line digital holography setup is used, an interference pattern arises since parts of the at least one object beam is scattered by the sample and interfere with those parts of the at least one object beam that are not scattered. In such a setup, it is preferable if the at least one translucent biological object only scatters light to a low degree as this improves the accuracy of the measurements. In an alternative setup, here referred to as off-axis digital holography, the interference pattern is obtained by combining the at least one object beam with at least one reference beam. Therefore, the apparatus may further comprise a beam dividing means arranged to divide the at least one beam into the at least one object beam and at least one reference beam, and wherein the sensor, the beam directing means, the beam dividing device, and the first reflecting surface are arranged relatively each other such that, when in use, the at least one reference beam and the at least one object beam that has passed through the sample are superposed at the sensor, thereby creating an interference pattern at the sensor. With this arrangement, a flexible and easy setup is obtained. The sample holder may present a first holding means having a first inner surface and a first outer surface, and a second holding means comprising said first reflecting surface, wherein said first inner surface and said first reflecting surface are provided to keep said sample and to be in contact with said sample. In accordance with the above, the noise may with this kind of arrangement be reduced due to reduced scattering of the at least one object beam. Preferably, the sample holder is detachable from said apparatus such that it conveniently may be replaced by a new sample in a new sample holder.

The first reflecting surface may have a coating for enabling cell growth.

In this way, cells may be grown directly on the first reflecting surface and may, for example, be studied and analyzed during different stages of the cell cycle. Further, the reflection coefficient of the first reflecting surface may be essentially equal to one. In this way, the incident at least one object beam and the reflected at least one object beam will have essentially the same intensity. Accordingly the signal detected at the sensor will be as strong as possible, thereby further improving the signal-to-noise ration and the accuracy of the hologram. The first reflecting surface may be formed from a metal coating or a dielectric coating. A dielectric coating is a coating whose refractive index and thickness are matched to the wavelength of the incident light, such that total reflection is obtained at the upper surface of the coating.

The sample holder may further comprise a pattern for identification of the sample holder. The pattern, which typically is unique for the sample holder, may be read by the apparatus and may for instance comprise information about the sample holder and the sample comprised in the sample holder.

In general, the pattern may be located anywhere on the sample holder. Preferably, however, the pattern is located at one of the first inner surface and the first reflecting surface. Further, the pattern may have a predetermined height and refractive index. In this way, the pattern may be used for calibration purposes. In particular, if the sample comprises a medium, such as air or a cell-growing fluid, the refractive index of the medium may be measured by means of the pattern. Moreover, the pattern may be used for calibration with respect to position, focus, degree of amplification, optical aberrations and phase bending.

The apparatus may further comprise a plurality of light sources, wherein the plurality of light sources create light of different wavelengths. It may be advantageous to use light of different wavelengths since the at least one biological object under study may respond differently to light of different wavelengths. Hence, by combining the results obtained by using different wavelengths, more information and possibly also more accurate information about the at least one biological object may be obtained.

Further, the apparatus may comprise a second reflecting surface arranged to reflect the at least one reference beam, wherein the beam dividing means is the same as the beam directing means, and wherein the beam dividing means comprises a beam splitter which is arranged to divide the at least one beam into at least one object beam and the at least one reference beam, to direct the at least one object beam towards the first reflecting surface at an essentially perpendicular angle with respect to the first reflecting surface, and to direct the at least one reference beam towards the second reflecting surface at an essentially perpendicular angle with respect to the second reflecting surface, and wherein the beam splitter is arranged to direct the at least one object beam which has been reflected in the first reflecting surface and the at least one reference beam which has been reflected in the second reflecting surface towards the sensor. In this way, a single beam splitter may be used to divide and direct the light beams.

In one embodiment, the at least one light source and the second reflecting surface are oppositely arranged, wherein the first reflecting surface and the sensor are oppositely arranged, and wherein the beam splitter is arranged between the at least one light source and the second reflecting surface as well as between the sensor and the first reflecting surface. In this way, an easy and flexible setup is obtained.

In one embodiment the reflection coefficient of the first reflecting surface may be less than one, wherein the apparatus further comprises a second sensor which is arranged to detect a part of the at least one object beam, said part of the at least one object beam having been transmitted by said first reflecting surface. With this arrangement, an off-axis digital holography setup where at least one reference beam is used may be combined with an in-line digital holography setup and the advantages of both setups may hence be drawn advantage of. More specifically, since the first reflecting surface has a reflection coefficient which is less than one, the at least one object beam will partly be transmitted through the first reflecting surface after having passed through the sample. The interference pattern originating from the transmitted parts of the at least one object beam are then detected by the second sensor. In this way, since the interference pattern at the second sensor is formed from the at least one object beam without a separate reference beam, the interference pattern at the second sensor corresponds to an in-line digital holography setup. At the same time, parts of the at least one object beam are reflected at the first reflecting surface and are superposed with the at least one reference beam to generate an interference pattern at the sensor.

It may be advantageous if the optical path lengths traveled by the at least one object beam and the at least one reference beam, respectively, are essentially the same. If so, the coherence length of the at least one beam may be short, implying that a cheap light source may be used. By making at least one of the first and second reflecting surfaces movable with respect to the beam splitter the optical path lengths of the at least one object beam and the at least one reference beam may be adjusted to be essentially the same.

In an alternative embodiment, the first outer surface is semi-translucent and acts as the beam dividing means, wherein the first outer surface is arranged at an angle relatively the first reflecting surface, such that the first outer surface partly transmits the at least one beam, thereby forming the at least one object beam, and partly reflects the at least one beam, thereby forming the at least one reference beam. With this arrangement, no separate component, such as a beam splitter, has to be used in order to divide the at least one beam. Preferably, the degree of transmission and reflection in the first outer surface may be chosen by letting the first outer surface have a predetermined relation between transmission and reflection.

The apparatus may further comprise an optical read head comprising the at least one light source and the sensor, wherein the optical read head and the first reflecting surface are movable relatively each other, thereby enabling scanning of the sample arranged on the first reflecting surface. The optical read-head may be an optical pick-up. In this way, information about a large part of the sample may be obtained in an easy way.

It is noted that since the optical read head does not comprise any microscope lenses for which focus have to be found, the optical read head does not have to be movable in a vertical direction relatively the sample holder. Thus, it is enough that the optical read head and the first reflecting surface are movable relatively each other in a lateral direction.

According to a third aspect, there is provided a sample holder for digital holographic microscopy comprising a first holding means having a first inner surface and a first outer surface; and

a second holding means having a second inner surface;

wherein the first and second inner surfaces are provided to keep a sample comprising at least one translucent biological object and at least one medium and to be in contact with the sample,

and wherein the second inner surface is an essentially flat reflecting surface.

According to a fourth aspect, there is provided use of a sample holder according to the third aspect for digital holographic microscopy.

According to a fifth aspect, there is provided use of an apparatus according to the second according to the third aspect for digital holographic microscopy.

It is noted that the invention relates to all possible combination of features recited in the claims. The features and advantages of the first and second aspects generally apply to the third, fourth and fifth aspects.

Other objectives, features and advantages of the present invention will appear from the following detailed disclosure, from the attached claims as well as from the drawings.

Generally, all terms used in the claims are to be interpreted according to their ordinary meaning in the technical field, unless explicitly defined otherwise herein. All references to "a/an/the [element, device, component, means, step, etc]" are to be interpreted openly as referring to at least one instance of said element, device, component, means, step, etc., unless explicitly stated otherwise. The steps of any method disclosed herein do not have to be performed in the exact order disclosed, unless explicitly stated.

Brief description of the drawings

The above, as well as additional objects, features and advantages of the present invention, will be better understood through the following illustrative and non-limiting detailed description of preferred embodiments of the present invention, with reference to the appended drawings, where the same reference numerals will be used for similar elements, wherein:

Fig. 1 is a schematic illustration of a digital holographic microscopy apparatus according to an embodiment of the invention;

Fig. 2 is a schematic illustration of a sample holder according to an embodiment of the invention; Fig. 3 is a schematic illustration of a digital holographic microscopy apparatus according to an embodiment of the invention;

Fig. 4 is a schematic illustration of a sample holder according to an embodiment of the invention;

Figs 5a-b are schematic illustrations from a top view and a side view, respectively, of a sample holder according to an embodiment of the invention;

Fig. 6 is a block diagram of a method according to an embodiment of the invention. Detailed description of preferred embodiments

Fig. 1 illustrates an apparatus 1 a for analyzing a sample 10 according to an embodiment of the invention. The apparatus 1 a comprises at least one light source 3, a sensor 5, a beam splitter 7 acting as a a beam directing means as well as a beam dividing means, a sample holder 13, and a second reflecting surface 15. Further, the sample holder 13 comprises a first reflecting surface 9.

The light source 3 is arranged to create at least one beam 19 of coherent light. The beam 19 of coherent light may for example be a laser beam which originates from any kind of laser source, such as a diode laser emitting light at a wavelength of 635 nm. Here, only one light source is illustrated. In general, however, the apparatus 1 a may comprise several light sources which may be used simultaneously. Preferably, if several light sources are used, these create light of different wavelengths. This may be advantageous since the sample 10 may react differently to light of different wavelengths. Hence more information about the sample may be obtained by using a plurality of light sources 3 having different wavelengths.

The beam 19 of coherent light originating from the light source 3 is directed towards a beam splitter 7. The beam splitter 7 divides the beam 19 of coherent light into an object beam 21 and a reference beam 23, thus acting as a beam dividing means. With this construction, the object beam 21 and the reference beam 23 become mutually coherent, implying that they have the same frequency and exhibit a constant phase relationship during the course of time. In case more than one light source 3 is present, the beam splitter 7 may divide each of the beams 19 originating from the light sources 3 into a mutually coherent object beam 21 and a reference beam 23.

As the beam 19 of coherent light incides on a beam dividing surface 8 of the beam splitter 7, parts of the beam 19 is transmitted and parts of the beam 19 is reflected. In the illustrated example, the part of the beam 19 that is transmitted through the beam dividing surface 8 forms the reference beam 23, and the part of the beam 19 that is reflected by the beam dividing surface 8 forms the object beam 21 . However, in an alternative embodiment, the transmitted beam may form the object beam 21 and the reflected beam may form the reference beam 23. Further, the beam dividing surface 8 of the beam splitter 7 is arranged relatively the light source 3 such that the angle of incidence of the beam 19 originating from the light source 3 is essentially 45 degrees, implying that the angle between the incident beam 19 and the reflected beam, here the object beam 21 , is essentially 90 degrees.

The sample holder 13 comprises a first reflecting surface 9 which is arranged to reflect the at least one object beam 21 . More precisely, the sample holder, and thereby the first reflecting surface 9, is arranged relatively the beam splitter 7 such that the object beam 21 is directed towards the first reflecting surface 9 at an angle which is essentially perpendicular to the first reflecting surface 9. In other words, the angle of incidence of the object beam 21 with respect to a normal of the reflecting surface is essentially zero degrees. The first reflecting surface 9 is essentially flat, implying that the laws of regular reflection known from fundamental physics applies to the first reflecting surface 9. Moreover, the coefficient of reflection of the first reflecting surface 9 is essentially equal to 1 , implying that the intensity of the reflected beam is essentially equal to the intensity of the incident beam.

When the apparatus 1 a is in use, a sample 10 to be analyzed is arranged on the first reflecting surface 9 of the sample holder 13. The sample may comprise at least one translucent biological object 1 1 . By translucent biological object 1 1 is meant a biological object 1 1 through which light may be transmitted. However, the biological object 1 1 may comprise absorbing parts, such as organelles. Examples of biological objects that may be analyzed are cells, ova, embryos, pollen grains, sperms, slide cultures, tissue sections or biopsy samples. Further, the sample may comprise at least one medium, such as a fluid. The fluid may be air or a cell culture fluid.

As the object beam 21 incides towards the first reflecting surface 9, the object beam 21 will pass through the sample 10 and, in particular, through the at least one biological object 1 1 prior to being reflected in the first reflecting surface 9. Since the object beam 21 passes through the sample 10, it will travel a longer optical path length compared to a beam, such as the reference beam 23, which does not pass through the sample, due to differences in refractive index. This will in turn lead to a phase shift between the object beam 21 and a beam which does not travel through the sample 10. The optical path length is defined as the physical/geometrical thickness multiplied with the refractive index.

When the object beam 21 has passed through the sample 10 it will be reflected in the first reflecting surface 9, and the direction of the reflected object beam 21 will be essentially opposed to the direction of the incident object beam 21 . Having been reflected, the object beam 21 will again pass through the sample 10 and hence again experience a longer optical path in comparison to the reference beam 23. As a result, the phase shift between the object beam 21 is twice the phase shift of a beam which only passes once through the sample. In particular, the phase shift obtained with the apparatus 1 a is twice the phase shift obtained in a digital holography apparatus which relies on a transmission principle where the corresponding object beam only passes once through the sample.

The second reflecting surface 15 is arranged to reflect the reference beam 23, or reference beams if there are more than one light source 3. More precisely, the second reflecting surface 15 is arranged opposite to the light source 3, and the beam splitter 7 is arranged between the light source 3 and the second reflecting surface 15. With this arrangement, the reference beam 23 incides on the second reflecting surface 23 when the apparatus 1 a is in use. Further, the second reflecting surface 15 is arranged relatively the beam splitter 7 such that the angle of incidence of the reference beam 23 with respect to a normal of the second reflecting surface 15 is essentially zero degrees. In this way, the second reflecting surface 15 is arranged to essentially oppose the direction of the reference beam 23 and redirect it towards the beam splitter 7. Preferably, the second reflecting surface 15 is essentially flat, thereby implying that the laws of regular reflection apply to the second reflecting surface 15. Moreover, the coefficient of reflection of the second reflecting surface 15 is preferably essentially equal to one.

As a skilled person readily understands, a similar apparatus is obtained by interchanging the positions of the sample holder 13 comprising the first reflecting surface 9 and the second reflecting surface 15.

As illustrated in Fig .1 , the second reflecting surface 15 is preferably movable with respect to the beam splitter 17 such that it may be moved towards or away from the beam splitter in a horizontal direction. In this way, the optical path length of the reference beam 23 relatively the optical path length of the object beam 21 may be conveniently adjusted in order to calibrate the apparatus 1 a. Alternatively, or additionally, the sample holder 13 may be movable with respect to the beam splitter 7.

The sensor 5, when in use, is arranged to detect an interference pattern arising from the object beam 21 and the reference beam 23. Here, the sensor 5 is arranged opposite to the first reflecting surface 9, and the beam splitter 7 is arranged between the sensor and the first reflecting surface 9. As the object beam 21 and the reference beam 23 have been reflected by the first and second reflecting surfaces 9 and 15, respectively, the beam splitter 7 superposes the object beam 21 and the reference beam 23 and directs the superposed beam 25 towards the sensor 5. As the object beam 21 and the reference beam 23 are mutually coherent they will generate an interference pattern at the sensor 5. The interference pattern is indicative of the phase shift between the object beam 21 and the reference beam 23. The sensor 5 may for example be a digital sensor such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor) image sensor.

The sensor may further be connected to a processing device (not shown) which may comprise software and/or hardware for carrying out any common reconstruction process. The reconstruction process may reconstruct phase and/or amplitude information from the interference pattern detected by the sensor 5. The reconstructed information may for example be used to obtain an image of the studied at least one biological object 1 1 . The information may for example also be used to determine the shape and optical density of the at least one biological object 1 1 .

In the illustrated example, the light source 3, the sensor 5, the beam splitter 7 and the second reflecting surface 15 are comprised in an optical read head 17. The optical read head 17 is preferably movable with respect to the sample holder 13 comprising the first reflecting surface 9. In this way, the object beam 21 will scan over the sample 10 and the at least one biological object 1 1 as the optical read head 17 is moved, and hence different parts of the sample 10 and the at least one biological object 1 1 may be analyzed. Alternatively, the sample holder 13 may be movable with respect to the optical read head 17.

It is noted that all optics is included in the optical read head 17, thus making the optics less sensitive to vibrations. Further, as the optical read head 17 is provided as one unit with fixed optics components it becomes easy to move relatively the sample holder 13. Moreover, the optical read head only comprises standard components, thereby making it cheap to produce. Further, the optical read head is similar to an optical read head, known as an optical pick-up, of a DVD- or blue ray-player. Hence, technology known from that area may be applied also in this case.

As a skilled person readily understands, the arrangement of the apparatus 1 a of Fig. 1 may easily be modified to an arrangement for in-line digital holography, wherein the hologram is created without using a reference beam. More precisely, such an arrangement may be achieved by arranging the beam splitter 7 such that it does not divide the beam 19, that is, such that the beam splitter 7 does not create a reference beam 23. In this way the beam splitter 7 will only act as a beam directing means and not as a beam dividing means. Further, the second reflecting surface 15 may be removed in such an arrangement.

Further, the arrangement of the apparatus 1 a of Fig. 1 may be modified to combine in-line digital holography and off-axis digital holography using a separate reference beam. More precisely, the first reflecting surface 9 may be semi-transparent, i.e. the reflection coefficient of the first reflecting surface 9 may be less than one. Similarly to the embodiment shown in Fig. 1 , parts of the at least one object beam 21 will then be reflected in the first reflecting surface 9 and be superposed with the at least one reference beam 23 to produce an interference pattern which is detected at the sensor 5. This corresponds to off-axis digital holography using a separate reference beam. However, in addition, parts of the at least one object beam 21 will be transmitted by the first reflecting surface 9. The parts of the at least one object beam 21 that are transmitted may be detected by a second sensor (not shown). For example, the second sensor may be located below the sample holder 13 of Fig. 1 . Due to the fact that parts of the at least one object beam 21 are scattered by the sample 10 before being transmitted, and parts of the at least one object beam 21 are not scattered before being transmitted, the second sensor will detect an interference pattern. This corresponds to in-line digital holography.

By varying the reflection coefficient of the first reflecting surface 9, the intensity of the interference patterns at the sensor 5 and the second sensor may be varied. In this way, the balance between in-line digital holography and off-axis holography using a separate reference beam may be adjusted. If the reflection coefficient is set to approximately 0.5, half the intensity of the at least one object beam 21 is transmitted by the first reflecting surface 9, and half the intensity of the at least one object beam 21 is reflected by the first reflecting surface 9.

In order to scan the sample 10 with this combined arrangement, the optical read head 17 and the second sensor may be moved simultaneously. Alternatively, the sample holder may be moved relatively the optical read head 17 as well as the second sensor.

Fig. 2 illustrates a sample holder 13a which may be used with the apparatus of Fig. 1 a. The sample holder 13a is arranged to hold a sample 10 comprising at least one translucent biological object 1 1 . The sample holder 13a comprises a first holding means 27 having a first outer surface 31 and a first inner surface 33, and a second holding means 29. In this case, the inner surface of the second holding means 29 is the first reflecting surface 9. The first reflecting surface 9 is essentially flat and has a reflection coefficient which is essentially equal to one. The first reflecting surface 9 may be formed from a metal coating or a dielectric coating. The dielectric coating is arranged to have a refractive index and thickness that matches the wavelength of the object beam 21 , such that total reflection is obtained as the object beam 21 incides on the first reflecting surface 9.

The first inner surface 33 and the first reflecting surface 9 are provided to keep the sample 10 and to be in contact with the sample 10. In this way, there will be no free surface of the sample which, due to vibrations, may be non-flat and hence scatter the object beam and cause noise. Preferably, the at least one translucent biological object 1 1 is located on the first reflecting surface 9. In the illustrated example, the first and second holding means 27 and 29 are essentially parallel to each other. In particular the first outer surface 31 is parallel to the first reflecting surface 9.

Further, in case the at least one biological object comprises biological cells, the first reflecting surface 9 may be provided with a coating that enables cell growth. Thus, at least one biological object may be grown on the first reflecting surface 9 of the sample holder 13a. Such a coating may be provided by treating the first reflecting surface 9 with a treatment to promote attaching of biological objects, such as cells. The treatment may be achieved by enhancing the cell affinity by coating the first reflecting surface 9 with a positively charged polymer, such as poly-lysine, or exposing the first reflecting surface 9 to a plasma treatment.

The first holding means 27 may be made of glass. Advantages with glass are that it normally is manufactured without any specific orientation of its components and thereby the glass is non-polarizing. In addition, it is possible to manufacture glass with very flat surfaces. In this way the scatter of the at least one object beam 21 when passing through the first holding means made of glass will be further reduced. Thereby, the quality of the hologram and the accuracy of the phase and amplitude information are further increased.

The sample holder 13a may comprise a box, preferably in the form of a cuboid or a cylinder, in which the sample 10 is kept. The first and second holding means 27 and 29 are then two opposite sides of the box. For example, if the at least one object beam 21 is incident from above, the first holding means 27 is the top side of the box and the second holding means 29 is the bottom side of the box.

Preferably, the sample holder 13a is provided as a separate unit which is detachable from the apparatus 1 a of Fig.1 . When used with the apparatus 1 a, an object beam 21 which incides towards the first reflecting surface 9 will, in order, pass through the first outer and inner surfaces 31 and 33 of the first holding means 27, and the sample 10 including the at least one biological object 1 1 prior to hitting the first reflecting surface 9. After reflection in the first reflecting surface 9, the object beam 21 will pass through the sample holder 13a in the reverse direction. Since the object beam 21 passes through the sample 10 twice it will be subject to a double phase shift as compared to if it only had passed only once.

Fig 3 illustrates an alternative apparatus 1 b for analyzing a sample 10 according to an embodiment of the invention. The apparatus 1 b comprises at least one light source 3, a sensor 5, a beam directing means 6, and a sample holder 13 comprising a first reflecting surface 9.

The at least one light source 3 is of similar type as the light source in the apparatus 1 a of Fig. 1 . The at least one light source 3 is arranged to create at least one beam 19 of coherent light which may be used to analyze a sample 10 comprising at least one biological object 10 arranged in the sample holder 13.

The light source 3 is arranged to direct the beam 19 towards the first reflecting surface 9 of the sample holder 13 via the beam directing means 6. The beam directing means 6 may for example comprise a mirror which reflects the beam 19. The beam directing means 6 may further be semi- transparent, thus not only reflecting light but also transmitting light. By varying the position and direction of the beam directing means 6, the beam 19 may be directed towards the first reflecting surface 9 at a desired angle.

Preferably, the position and direction of the beam directing means 6 is arranged to direct the beam 19 towards the first reflecting surface 9 of the sample holder 13 in a direction which is essentially perpendicular to the first reflecting surface 9.

The sample holder 13 is arranged to hold a sample 10 comprising at least one biological object 1 1 . As will be explained in more detail below with reference to Fig. 4, the sample holder comprises a first outer surface 31 which is arranged above the sample 10 and the first reflecting surface 9, and at an angle relatively the first reflecting surface 9. Further, the first outer surface 31 is semi-translucent. For example, the first outer surface 31 may be treated to obtain a predetermined relationship between transmission and reflection. Consequently, as the at least one beam 19 incides towards the sample holder 13, parts of the at least one beam 19 is transmitted by the first outer surface 31 , thereby forming at least one object beam 21 , and parts of the at least one beam 19 is reflected by the first outer surface 31 , thereby forming at least one reference beam 23. Hence, the first outer surface 31 acts as a beam dividing means which divides the at least one beam 19 into at least one object beam 21 and at least one reference beam 23.

The at least one object beam 21 , which has been transmitted by the first outer surface 31 , passes through the sample 10, is reflected at the first reflecting surface 9, and passes through the sample again in an essentially reversed direction. In this way, the at least one object beam 21 will experience a phase shift relatively the reference beam 23. In particular, the phase shift is twice the phase shift of a light beam that only passes the sample 10 once.

The sensor 5 of apparatus 1 b is of a similar type as the sensor of apparatus 1 a of Fig .1 . In the example of Fig. 3, the sensor 5 and the first reflecting surface 9 are essentially oppositely arranged. With this

arrangement, the at least one object beam 21 that has passed twice through the sample 10 will incide on the sensor 5. Further, the position and

dimensions of the sensor 5 are such that also the at least one reference beam 23 which is reflected from the first outer surface 31 incides on the sensor, thereby generating an interference pattern at the sensor. As is shown in this schematic example, the at least one reference beam 23 and the at least one object beam 21 do not incide at the same point on the sensor 5 since the beams are not parallel as a result of the inclination of the first outer surface 31 . This may be advantageous when reconstructing phase and/or amplitude information from the interference pattern.

The at least one light source 3, the sensor 5 and the beam directing means 6 may be part of an optical read head 17. As previously disclosed the optical read head 17 and the sample holder 13 may be movable relatively each other.

As is understood by a person skilled in the art, there are modifications of the apparatus 1 b which would lead to a similar result and which falls within the scope of the invention. For example, there are many possible variations of the positions of the light source 3, the sensor 5 and the beam dividing means 6. For example, the light source 3 may be arranged essentially oppositely the first reflecting surface 9, such that the at least one beam 19 does not have to be redirected by the beam directing means prior to being reflected by the first reflecting surface 9. Instead, the beam directing means 6 may be used to direct the at least one object beam 21 that has passed through the sample 10 twice and/or the reference beam 23 towards the sensor 5, wherein the sensor 5 and the first reflecting surface 9 are not oppositely arranged.

Fig. 4 illustrates a sample holder 13b which may be used in connection to the apparatus 1 b of Fig. 3. In most respects, the sample holder 13b is similar to the sample holder 13a of Fig. 2. However, an important difference is that in sample holder 13b the first holding means 27 is arranged at an angle a relatively the second holding means 29. In particular, the first outer surface 31 is arranged at an angle a relatively the first outer surface 31 . The angle a is typically small, in the order of magnitude of one degree. However, the optimal angle depends on various parameters of the setup of the apparatus 1 b, such as the distance between the sample holder 13 and the sensor 5. Moreover, the first outer surface 31 is semi-translucent. For example, it may be treated to have a desired relation between transmission and reflection. In this way, the first outer surface 31 may act as a beam dividing means for an incident light beam, such as the at least one beam 19. As illustrated in the example, the beam 19, which is essentially perpendicular to the first reflecting surface 9, is partly transmitted by the first outer surface 31 , thereby forming the object beam 21 , and partly reflected by the first outer surface 31 , thereby forming the reference beam 23. Due to the inclination with angle a of the first outer surface 31 relatively the first reflecting surface 9, the angle between the object beam 21 and the reference beam 23 is 2a. Figs 5a and 5b illustrate a sample holder 13c comprising a pattern 39 which may be used for identification and calibration. The basic design of the sample holder 13c may be any of the designs previously described, including the designs of sample holders 13a and 13b. However, in addition, the sample holder 13c comprises a pattern 39. The pattern may correspond to a unique code which may be decoded by the apparatus of the invention. Thereby, the apparatus may identify the sample holder 13c. Based on the unique code, information concerning the sample holder 13c may be inserted into a data base. For example, the information may comprise time points when the sample was analyzed, type of sample, previous measurements on the sample, measurement results, position of the sample holder etc. The pattern may for example be a grid, e.g. a grid of lines or dots.The pattern may additionally or alternatively be designed to have a predetermined thickness d and refractive index n 1 . If so, it may be used to determine a refractive index n2 of the medium of the sample by measuring a relative phase shift according to the formula

where APS is a relative phase shift and _4n=n1 -n2. When determining n2, the relative phase shift APS, the refractive index n1 of the pattern, and the thickness d are known. In this way, the pattern may be used to calibrate the apparatus according to the invention. As shown in Fig. 5b, the pattern may be located at the first reflecting surface 9. Alternatively, it may be located at the first inner surface. The calibrating pattern 39 may be formed during the manufacturing of the sample holder, e.g. by using a mould with a recess, or by fixing an object to the sample holder 13c.

A method according to an embodiment of the invention will now be described with reference to Fig. 6 and Figs 1 -4. The boxes in the flow chart drawn with a dashed line are optional steps. The steps of the method do not have to be carried out in any particular order unless explicitly stated.

In a step S602, a sample 10 comprising at least one translucent biological object 1 1 is arranged on a first reflecting surface 9 of a sample holder 13. For example, the sample 10 may be arranged on the first reflecting surface of sample holder 13a, 13b or 13c.

In step S604, at least one beam 19 of coherent light is created. For example the at least one beam 19 may be created by at least one light source 3. If more than one beam is created, these preferably have different wavelengths. The at least one beam 19 comprises at least one object beam 19. This is to be interpreted in the sense that at least parts of the at least one beam 19 may be used as at least one object beam 21 .

In step S606, the at least one beam 19 may be divided into at least one object beam 21 and at least one reference beam 23. The at least one object beam 21 and the at least one reference beam 23 will in this way be mutually coherent. As shown in Fig. 1 , the at least one beam 19 may be divided by means of a beam splitter 7. Alternatively, and as shown in Fig. 3, the at least one beam 19 may be divided by means of a first outer surface 31 of a sample holder 13. In general, the at least one object beam 21 is divided by a beam dividing device.

In step S608, the at least one object beam 21 is directed towards a first reflecting surface 9 of a sample holder 13. Specifically, the at least one object beam 21 is directed towards the first reflecting surface 9 at an angle which is essentially perpendicular to the first reflecting surface 9. The at least one object beam 21 may be directed towards the first reflecting surface 9 by arranging the light source 3 essentially opposite to the first reflecting surface 9 or by directing it via a beam directing device. For example, in Fig. 1 , the at least one object beam 21 is directed towards the first reflecting surface 9 by a beam splitter 7, and in Fig. 3, the at least one object beam 21 is directed by using a beam directing device 6. Note that the in Fig. 1 the at least one object beam 21 is directed after dividing the at least one beam 19, whereas in Fig. 2 the at least one beam 19, and thereby indirectly the at least one object beam 21 , is directed prior to dividing the at least one beam 19.

A result of directing the at least one object beam 21 towards the first reflecting surface 9at an angle which is essentially perpendicular to the first reflecting surface 9 is that the at least one object beam 21 passes through the sample in a first direction, is reflected by the reflecting surface 9, and passes through the sample in a second direction which is essentially opposed to the first direction. In other words, the at least one object beam 21 passes twice through the sample 10.

In step S610, the at least one object beam 21 and the at least one reference beam 23 are superposed. In the embodiment of Fig. 1 , a second reflecting surface 15 and the beam splitter 7 are used to superpose the at least one object beam 21 and the at least one reference beam 23.

Specifically, the second reflecting surface 15 is used to reflect and direct the at least one reference beam 23 towards the beam splitter 7 where it is superposed with the at least one object beam 21 and directed towards the sensor 5. In the embodiment of Fig. 3, the sensor 5 is arranged relatively the sample holder in such a way that the at least one object beam 21 and the at least one reference beam 23 are superposed at the sensor 5. As a result, an interference pattern will be generated at the sensor 5.

In step S612, the interference pattern originating from the at least one object beam and, if present, the at least one reference beam 23 is detected. Step S612 is typically detected by a sensor 5 as previously described.

In step S614, phase and/or amplitude information is reconstructed from the detected interference pattern. Step S614 is typically carried out by a processing unit comprising hardware or software instructions pertaining to a reconstruction method. The reconstructed information may for example be used to obtain an image of the studied at least one biological object. The information may for example also be used to determine the shape and optical density of the at least one biological object.

In case the steps of the dashed boxes of Fig. 6 are not carried out, a method for analyzing a sample comprising at least one translucent biological object by means of an in-line digital holographic microscopy is obtained.

The invention has mainly been described above with reference to a few embodiments. However, as is readily appreciated by a person skilled in the art, other embodiments than the ones disclosed above are equally possible within the scope of the invention, as defined by the appended patent claims.