Device, method and computer program

Abstract

A device, the device being configured to determine multi-point refocusing information using a target (60, 95) to realize object plane (13)- and / or sensor plane (31)- calibration in a holographic microscope (1).

Description

DEVICE, METHOD AND COMPUTER PROGRAMTECHNICAL FIELDThe present disclosure generally pertains to the field of holographic microscopy, in particular to devices, methods and systems for time-sequential partially coherent illumination based holographic microscopy scanners.TECHNICAL BACKGROUNDHolographic microscopy (e.g., digital in-line holographic microscopy, DIHM) is digital holography applied with regards to microscopy. Holographic microscopy scanners distinguish themselves from other microscopy methods by not directly recording the projected image of an object but instead by recording a hologram. The hologram (also called hologram intensity plane) is recorded by a digital image sensor or by a photodetector.Based on the recorded hologram, the complex field information, that is the corresponding phase and amplitude image of the scanned object, is determined by means of a numerical reconstruction algorithm (e.g., iterative projection). An image forming lens in traditional microscopy is thus replaced by the numerical reconstruction algorithm.It is beneficial if an object-detector distance is known precisely for the numerical reconstruction algorithms to work properly. Therefore, prior to reconstruction, it is necessary to estimate the object-detector distance, a process which is also called refocusing. Improper refocusing on hologram intensity planes may cause outliers leading to complete loss of information of individual hologram intensity planes. Further, refocusing errors may be caused by noisy hologram intensity planes and diverging refocusing results may be obtained in case of multiwavelength acquisitions.Therefore, it is desirable to improve the refocusing within a digital in-line holographic microscopy.SUMMARYAccording to a first aspect, the disclosure provides a device being configured to determine multipoint refocusing information using a target to realize object plane- and / or sensor planecalibration in a holographic microscope.According to a further aspect, the disclosure provides a method comprising determining multipoint refocusing information using a target to realize object plane- and / or sensor planecalibration in a holographic microscope.Further aspects are set forth in the dependent claims, the following description and the drawings.BRIEF DESCRIPTION OF THE DRAWINGSEmbodiments are explained by way of example with respect to the accompanying drawings, in which:Fig. 1 schematically shows a digital in-line holographic microscopy (DIHM) scanner in which a phase shift between interference images is realized by changing the object-to-sensor distance;Fig. 2 shows an embodiment of a classification process performed on an input sample;Fig. 3 schematically shows an example of a digital in-line holographic microscope (DIHM) scanner that provides hardware object plane- and / or sensor plane-calibration;Fig. 4a schematically shows in more detail the rotation platform configured to control the yaw angle of the object plane of the in-line holographic microscopy (DIHM) scanner;Fig. 4b schematically illustrates the yaw angle that the rotation platform in Fig. 4a is configured to control;Fig. 5a schematically shows in more detail the rotation platform configured to control the pitch angle of the object plane of the in-line holographic microscopy (DIHM) scanner;Fig. 5b schematically illustrates the pitch angle that the rotation platform in Fig. 5a is configured to control;Fig. 6 shows an embodiment of a 9-point phase calibration target for object plane- and / or sensor plane-calibration in a digital in-line holographic microscope (DIHM) system;Fig. 7 shows an example measurement of the focus distance for the 9-point refocus grid of Fig. 6 based on the Sobel magnitude metric;Fig. 8 shows a 3D plot of the focus distances determined in Fig. 7 for the 9-point refocus grid;Fig. 9 shows a flow diagram of the multi-point refocusing process to realize object plane- and / or sensor plane-calibration in a digital in-line holographic microscope (DIHM);Fig. 10 schematically shows exemplary components of a digital in-line holographic microscope (DIHM) to implement a multi-point refocusing process for object plane- and / or sensor planecalibration;Fig. I la shows an exemplary user interface element generated by a user interface;Fig. 1 lb shows another exemplary user interface element generated by a user interface;Fig. 11c shows another exemplary user interface element generated by a user interface;Fig. l id shows another exemplary user interface element generated by a user interface;Fig. 12a shows a surface-functionalized microscope slide in a top-view;Fig. 12b show a surface-functionalized microscope slide in a cross-sectional view;Fig. 13 shows a computer-implemented process of automated pitch and yaw correction; andFig. 14 schematically describes an embodiment of an electronic device which may implement the functionality of the process steps described herein.DETAILED DESCRIPTION OF EMBODIMENTSBefore a detailed description of the embodiments under reference of Fig. 1, general explanations are made.The embodiments described below in more detail disclose a device being configured to determine multi-point refocusing information using a target to realize object plane- and / or sensor plane-calibration in a holographic microscope.The holographic microscope may for example be a digital in-line holographic microscope (DIHM).A DIHM system captures intensity hologram planes at certain object-to-sensor distances.This may provide improved quality and accuracy of the phase and amplitude reconstruction over the complete acquisition stack across the entire field-of-view (FoV), in particular for multiwavelength systems.The multi-point refocusing may allow to detect alignment deviations from the ideal condition.The calibration as described above may for example be applied in the field of live cell imaging applications.The device may be configured to provide information about an orientation of an alignment plane.The alignment plane may for example relate to the object and / or sensor plane. For example, the alignment plane may describe the orientation of the object plane in the coordinate system of the sensor plane. Alternatively, the alignment plane may describe the orientation of the sensor planein the coordinate system of the object plane. This may allow to detect misalignment between the object and sensor plane.According to some embodiments, the device may further be configured to adjust the orientation of the alignment plane.For example, the device may provide the possibility of rotating the object plane to achieve alignment of the object plane and the sensor plane. Alternatively, the device may provide the possibility of rotating the sensor plane to achieve alignment of the object plane and the sensor plane.The quality of the reconstruction may be improved by means of aligning object and / or sensor plane parallel to each other, such that the object-to-sensor distance is the same for all points in the captured hologram.Thus, it is possible to detect pitch and yaw angle of said adjustment plane, e.g., caused by a misalignment between object and sensor plane.According to some embodiments, the device may further be configured to provide for pitch and yaw correction.The device may further comprise a pitch rotation platform and a yaw rotation platform to provide pitch and yaw correction.According to some embodiments, the device may be configured to provide n-point refocusing information obtained from a phase image of a phase target.According to some embodiments, the device may be configured to apply the Sobel magnitude criterion for obtaining the refocusing information.According to some embodiments, the device may comprise a calibration target with multiple refocusable structures, where the locations of the refocusable structures span a 2D plane in 3D space.This 2D plane may be considered as an adjustment plane, based on which the orientation of the object plane with regard to the sensor plane can be determined. This adjustment plane can be aligned, e.g., such that the object plane becomes parallel to the sensor plane and all n refocusing points have the same object-to-sensor distance.The calibration target may be a phase calibration target.According to some embodiments, the calibration target may use polystyrene (PS) beads as refocusing targets.The size of the beads, their size distribution, and their density on surface may be tailored to fit the respective requirements.According to some embodiments, the calibration target may comprise a slide surface- functionalized in a way that the cover glass contains covalently attached polystyrene beads.The polystyrene beads may have a diameter of 5 pm.Further, the polystyrene beads may be provided with carboxyl functional groups at their surface.Still further, the polystyrene beads may be configured to form a monolayer structure.For example, all polystyrene beads may reside in the same plane.According to some embodiments, the slide may be densely covered with polystyrene beads.According to some embodiments, the polystyrene beads may be of substantially equal size.If substantially equally sized PS particles cover the entire FoV, calibration may for example be performed in a "one-shot" -approach, requiring only a single image to be acquired. This may avoid having spread features of different dimension across the target so that the target does not need to be moved across the FoV (thus limiting accuracy to the stage movement accuracy). This may also avoid empirical correction of the size variation.Further, a polystyrene bead target as described in the embodiments is cost efficient, so that a polystyrene bead target may be delivered with each DIHM for customer's convenience.For example, the size distribution of the polystyrene beads may be such that the coefficient of variation CV is smaller than 3%. For example, the coefficient of variation CV may be within 1- O / / O.The specially tailored target for refocusing is easily detectable, as it may come, e.g., as separate item delivered with the main device. The opto-mechanic part of our proposed solution is detectable based on the devices necessary for adjustment of yaw or pitch in the object and / or sensor plane. To detect the algorithmic part of our proposed solution, deep knowledge of the system and / or firmware decoding is required.The embodiments described below in more detail further disclose a method comprising determining multi-point refocusing information using a target to realize object plane- and / or sensor plane-calibration in a holographic microscope.According to some embodiments, the method may comprise capturing an image of a calibration target with a holographic microscope.According to some embodiments, the method may comprise obtaining n-point focus distances of phase points according to the Sobel magnitude criterion.According to some embodiments, the method may comprise determining an alignment plane from distances and positions of the phase points.The determining of the alignment plane may for example be realized by least square optimization.According to some embodiments, the method may comprise determining pitch and yaw corrections based on an orientation of the alignment plane.According to some embodiments, the method may comprise presenting pitch and yaw corrections to the user.According to another embodiment, the method may comprise automatically controlling pitch and yaw according to a determined pitch correction and a determined yaw correction.According to some embodiments, the method may comprise covering a polystyrene bead target by a fluidic cell or bringing it into the fluidic cell comprising a fluid with a given refractive index.By applying a fluid of chosen refractive index as the host medium for the polystyrene bead sample, tunability of the refractive index contrast between polystyrene beads and their environment may be achieved. This can be useful to avoid phase wrapping during the calibration process, in particular when calibrating the system at different wavelengths.According to another embodiment, the method may comprise changing the fluid during measurements to adapt it to the respective wavelength under use.According to some embodiments, the method may comprise changing the fluid during measurements to increase data diversity.The method may, for example, be a computer-implemented method. The embodiments also disclose a computer-readable medium comprising instructions to implement the methods described herein.The embodiments further disclose a phase calibration target for use in a multi-point refocusing process to realize object plane- and / or sensor plane-calibration in a holographic microscope (1).The phase calibration target may comprise multiple refocusable structures, where the locations of the refocusable structures span a 2D plane in 3D space.The embodiments further disclose a calibration target comprising polystyrene beads as refocusing targets.According to some embodiments, the polystyrene beads span a 2D plane in 3D space.According to some embodiments, the calibration target comprises a slide surface-functionalized in a way that the cover glass contains covalently attached polystyrene beads.The polystyrene beads may have a diameter of 5 pm.The polystyrene beads may be provided with carboxyl functional groups at their surface.According to some embodiments, the polystyrene beads may be configured to form a monolayer structure.According to some embodiments, the slide may be densely covered with polystyrene beads.According to some embodiments, the polystyrene beads may be of substantially equal size.For example, the size distribution of the polystyrene beads may be such that the coefficient of variation CV is smaller than 3%.Embodiments are now described with reference to the drawings.Digital in-line holographic microscopy (DIHM)In the following embodiments, a computational imaging system, in particular a digital in-line holographic microscope scanner as described in more detail in international patent application WO 2022 / 008726 Al is applied in the field of digital pathology imaging (DPI) and is used in order to create an image of an object, for example a sample (e.g. a body sample, a fluidic sample or the like), and to perform classification processing of the image.Fig. 1 schematically shows a digital in-line holographic microscopy (DIHM) scanner 1 in which a phase shift between interference images is realized by changing the object-to-sensor distance. An in-line holography laser 8 emits partially coherent time sequential illumination light beams 6 at different predefined illumination light wavelengths. An object plane 13 is located at a distance ZLO from the laser 8. Into this object plane 13, an object 12, for example a fluid cell, is placed. For each of the different predefined illumination light wavelengths, a monochrome image sensor 14, for example a CMOS or a CCD sensor, captures a respective interference pattern created by superposition of a wave front scattered (also called scattered wave) by the object 12 and a nonscattered wave front (also called reference wave). The non-scattered wave originates from the partially coherent illumination source. The monochrome image sensor 14 is placed at a distance zos from the object plane 13 in which the object 12 is located. In order to capture differentinterference patterns with different phase shifts, the monochrome image sensor 14 is shifted by an actuator such as a servomotor (not shown in Fig. 1) and is thus placed at specified positions 14A and 14B. Accordingly, the distance zos between the image sensor 14 and the object plane 13 is altered and different interference patterns can be recorded at the monochrome image sensor 14 with respective phase shifts.This setup, where the laser 8 sends its beams 6 directly through the object 12 onto the monochrome image sensor 14 where the interference pattern is captured, is called digital in-line holography. It has to be noted that in this setup no lens (lens-free) is used. In this case a pinhole may be added on the illumination side. The interference pattern is also called “hologram” (or “intensity image plane”), wherein each hologram encodes amplitude and phase information of the object 12. In order to realize a proper illumination, an aperture may be added to the configuration.In the embodiment of Fig. 1, a phase shift between interference images is realized by changing the object-to-sensor distance by an actuator such as a servomotor. It should however be noted that a phase shift between interference images may alternatively be realized with a tuneable phase-shifter. This has the advantage that no parts have to be moved inside the apparatus in order to obtain the phase shift and therefore the susceptibility to errors is further reduced. In yet other embodiments, a phase shift between interference images may be realized by inserting optical elements into the light path, or a phase shift between interference images may be realized with a switchable polarizer and a birefringent optical element.Digital processing of captured hologramsEach material in the light pass (e.g., a fluid, a microparticle, etc.) introduces a delay in the light path when light propagates through it. This amount of delay is also called “phase shift” or “Optical Path Difference”. The DIHM scanner and its operation as described in Fig. 1 above and in international patent application WO 2022 / 008726 Al allows to control / introduce extra phase shift when capturing interference images. In particular, for each of a set of predefined wavelengths, multiple interference images (holograms) are captured at different phases. In other words, for each wavelength, multiple interference images (holograms) are captured with predefined phase shifts.Fig. 2 shows a classification process performed on an input sample (body sample, fluidic sample, or the like). In process step 201, a sample is input into a DIHM scanner (1 in Fig. 1). In process step 202, a number of M phase-shifted holograms (here for example M=3) of the sample are taken per N different illumination wavelengths (here for example N=3), wherein the N differentillumination wavelengths are applied time-sequentially. This results in a total number of M x N holograms. In process step 203, for each different illumination wavelength a quantitative phase image (QPI) and an amplitude of the sample is reconstructed. In process step 204, the resulting quantitative phase images (QPIs) for each wavelength are collected in a QPI stack, the intensity images are collected in a colour image and a quantitative dispersion image (QDI) of the sample is constructed. In process step 205, a classifier is operated on the QDI.The different phase-shifted holograms captured by a DIHM scanner per wavelength contain amplitude and phase information related to the scanned sample (12 in Fig. 1) which is encoded into the interference patterns (holograms). For each illumination light wavelength, amplitude and phase information of the scanned sample 12 can be decoded by means of an algorithm such as for example the so called Gerchb erg- Saxton algorithm, which is for example explained in “A Practical Algorithm for the Determination of Phase from Image and Diffraction Plane Pictures” by W. Gerchberg and W. O. Saxton from 29 November 1971, or in the book “Handbook of Optical Systems: Metrology of Optical Components and Systems, Volume 5” by Herbert Gross, Bernd Dorband, Henriette Muller.In general, the Gerchberg-Saxton algorithm is an iterative algorithm which allows to retrieve the phase of a pair of light distributions / planes which stem from two operation modes - imaging and diffraction. The light distribution in the image plane (also called image plane wave) and the light distribution in the diffraction plane (also called diffraction plane wave) are related via a propagating function, such as the Fourier transform. The amplitude of the light distribution in the diffraction plane is given by the recorded hologram as described above. The two or more phase- shifted holograms (M holograms in Fig. 2) can be used in the algorithm to speed-up convergence. The amplitude of the image plane wave can be obtained by a Fourier transform of the diffraction plane wave as explained in more detail in WO 2022 / 008726 Al.Dispersion in optics describes the phenomenon that the refractive index of a material differs for different wavelength. Constructing a quantitative dispersion image of a sample allows to quantize the dispersion of each image pixel. As described in more detail in WO 2022 / 008726 Al, a quantitative dispersion value QDVsampleof the sample at a respective pixel of the image can be calculated according to:where ngreen, sample, nbiue, sample and nred, sample are the refractive indices of the sample for green, blue and red light respectively.By calculating this quantitative dispersion value QDVobjectfor each pixel, the quantitative dispersion image QDI is obtained.The above-described determination of the QDI can be done with any wavelength that the DIHM scanner operates with. The determination may for example be done for three illumination wavelengths. If, for example, three different wavelengths are applied, these three wavelengths may be ordered as: Xiong > middie >short, and with the corresponding refractive indices niong, nmiddie , nshort , the quantitative dispersion value QDVsamplefor each pixel can be calculated by:The same principles apply if less than three or more than three different wavelengths are applied.A quantitative dispersion image QDI is determined by calculating, for each pixel, a quantitative dispersion value QDVsamplebased on the respective refractive indices ni sampleof the wavelengths i.ClassificationThe quantitative dispersion image QDI is a fast and compact way to condense a “phase characteristic” of the scanned sample (12 in Fig. 1) into one image. This is especially useful if a classification and visualisation is done using a classification learning algorithm.As described in more detail in WO 2022 / 008726 Al, a classification learning algorithm can operate with the quantitative dispersion image QDI as such, or on a combination of the quantitative dispersion image QDI with QPI data and colour image data.Classification algorithms are known to the skilled person. For example, there exist classification algorithms that are based on supervised learning to obtain a trained classifier. For example, linear regression, linear classifiers (Naive Bayes, perceptron, logistic regression), quadratic classifiers, support vector machines (SVM), kernel density estimators, k-nearest neighbor, artificial neural networks (ANN) or more.Multi-point refocusingA DIHM system as described above captures intensity hologram planes at certain object-to- sensor distances. As described with regard to Fig. 2, to reconstruct complex field information, the corresponding phase and amplitude images at the object and / or detector are determined bymeans of reconstruction (e.g., iterative projection), which implies knowledge of the relative object-camera distance and orientation.To determine these relations and thus to improve the quality of reconstruction, the embodiments described below disclose a multi-point refocusing process to detect misalignment between object and sensor plane and a hardware calibration to adjust the object and / or sensor plane to achieve parallel alignment. The multi-point refocusing is configured to detect alignment deviations from the above-mentioned ideal condition and perform a calibration to adjust the object and / or sensor plane to achieve parallel plane alignment (e.g., by pitch and yaw correction). The quality of the reconstruction is improved by means of this aligning of object and / or sensor plane parallel to each other, such that the object-to-sensor distance is the same for all points in the captured hologram.Fig. 3 schematically shows an example of a digital in-line holographic microscope (DIHM) scanner that provides hardware object plane- and / or sensor plane-calibration. As indicated by an arrow 30, the digital in-line holographic microscopy (DIHM) scanner 1 realizes a phase shift between interference images by changing the object-to-sensor distance. An object is placed into the object plane 13 of the scanner 1. An in-line holography laser 8 directs partially coherent time sequential illumination light beams onto the object plane 13. In the sensor plane 31 of the scanner 1, an image sensor (14 in Fig. 1) is placed that captures images and sends them to a processor for further processing to obtain the QPI / QDI images. A rotation platform 33 is provided to configure the orientation of the object plane 13, in particular a yaw angle. A rotary control 32 is provided by which the yaw angle of the rotation platform 33 can be manually configured. A pitch platform 35 is provided to configure the pitch of the object plane 13. A rotary control 34 is provided by which the pitch angle of the rotation platform 35 can be manually configured. This hardware calibration allows to adjust the orientation of the object to achieve parallel alignment.Fig. 4a schematically shows in more detail the rotation platform 33 which is provided to configure the orientation of the object plane. Rotary control 32 is provided by which the user of the device may adjust the yaw angle of the rotation platform 33. A scale 37 is provided which allows the user to visually track changes of the yaw angle.Fig. 4b schematically illustrates the yaw angle that the rotation platform 33 in Fig. 4a is configured to control. The Xv-axis, the Yv-axis and the Zv-axis represent the coordinate system of the DIHM scanner 1. The Xv-axis, the Yv-axis describe the plane that is parallel to the ground. The Zv-axis describes the direction that is orthogonal to the ground (upward direction). As indicated by arrowA, the rotation platform 33 is provided to control the yaw angle, that is, a rotation around the Zv- axis. Ze indicates the orientation of the object plane in the coordinate system of the DIHM scanner 1.Fig. 5a schematically shows in more detail the pitch platform 35 which is provided to configure the pitch of the object plane. Rotary control 34 is provided by which the pitch angle of the pitch platform 35 can be manually configured. This hardware calibration allows to adjust the orientation of the object to achieve parallel alignment. A screw 38 is provided by which the configuration of the pitch platform 35 can be loosened, so that pitch control with rotary control 34 becomes accessible, and tightened, so that the pitch configuration, once properly selected, is locked.Fig. 5b schematically illustrates the pitch angle that the pitch platform 35 in Fig. 5a is configured to control. As in Fig. 4b, the Xv-axis, the Yv-axis and the Zv-axis represent the coordinate system of the DIHM scanner 1. The Xv-axis, the Yv-axis describe the plane that is parallel to the ground. The Zv-axis describes the direction that is orthogonal to the ground (upward direction). As indicated by arrow B, the pitch platform 35 is provided to control the pitch angle, that is, a rotation around the Yv-axis. Ze indicates the orientation of the object plane in the coordinate system of the DIHM scanner 1.In the embodiment of Fig. 3, the rotation platform 33 and the pitch platform 35 are provided to adjust the orientation of the object plane 13 to achieve parallel alignment with the sensor plane 31. In alternative embodiments, the hardware may be configured to adjust the orientation of the sensor plane 31 instead of that of the object plane, or, in yet other embodiments, both could be adjusted by respective controls.Multi-point calibrationIn the embodiments described below in more detail, a multi-point refocusing process to realize object plane- and / or sensor plane-calibration in a digital in-line holographic microscope (DIHM) system is disclosed. For example, a n-point refocusing process and a special calibration target with n refocusable structures is disclosed, where the n resulting refocusing points span a 2D plane (adjustment plane) in 3D space. With the calibration target, it is possible to detect a pitch and yaw angle of the adjustment plane (e.g. object plane or sensor plane), e.g., caused by a misalignment between object and sensor plane. The adjustment plane can be aligned according to the principles described above with regard to Fig. 3, 4a, b and 5a, b such that the object plane becomes parallel to the sensor plane and all n refocusing points have the same object-to-sensor distance.According to an embodiment, the calibration target corresponds to a phase calibration target as it is generally known to the skilled person. Phase calibration targets are for example described by T. M. Godden et al in “Phase calibration target for quantitative phase imaging with ptychography”, Optics Express Vol. 24, Issue 7, pp. 7679-7692 (2016). A quantitative phase calibration target is typically used to confirm calibration of a Quantitative phase imaging (QPI) product. A phase calibration target may for example be fabricated using reactive ion etching (RIE) of amorphous SiO? patterned via optical lithography. A phase calibration target may for example contain features etched 100 nm to 1000 nm deep into transparent amorphous SiCh and have feature sizes spanning a wide range of spatial frequencies - from length scales of 2 pm to length scales of 600 pm. The amorphous SiO2 may for example be grown on 1 optically flat quartz windows.Fig. 6 shows an embodiment of a 9-point phase calibration target that can be used to realize object plane- and / or sensor plane-calibration in a digital in-line holographic microscope (DIHM) system. 9 phase points PPI to PP9 are etched as features into a 2300 nm x 1700 nm transparent SiCE layer 60. The depth of each phase point PPI to PP9 is, in this example, chosen to be 600 nm. The structure size in the example is varying. For example, the diameter of each phase point may be chosen to be 100 pm.According to the embodiment described below in more detail, the calibration is based on a "one- shot" -approach, requiring only a single image of the calibration target to be acquired. The focus distance of each phase point PPI to PP9 is determined according to the Sobel magnitude metric.Fig. 7 shows an example measurement of the focus distance for a region of interest (ROI) of the 9-point refocus grid of Fig. 6 based on the Sobel magnitude metric. On the x-axis the phase points PPI to PP9 are represented by their respective number 1, 2, . . ., 9. On the y-axis, the focus distance in mm for each phase point as obtained according to the Sobel magnitude criterion is depicted. For phase point PPI a focus distance of 1.458 mm has been determined. For phase point PP2 a focus distance of 1.463 mm has been determined. For phase point PP3 a focus distance of 1.440 mm has been determined. For phase point PP4 a focus distance of 1.476 mm has been determined. For phase point PP5 a focus distance of 1.474 mm has been determined. For phase point PP6 a focus distance of 1.470 mm has been determined. For phase point PP7 a focus distance of 1.491 mm has been determined. For phase point PP8 a focus distance of 1.488 mm has been determined. For phase point PP9 a focus distance of 1.491 mm has been determined. In this example, the phase points PPI to PP9 have a spread of 51 pm.Fig. 8 shows a 3D plot of the focus distances determined in Fig. 7 for the 9-point refocus grid. The x-axis and the y-axis represent the position of the phase points PPI to PP9 in the target plane. On the z-axis, the focus distance in mm for each phase point as obtained according to the Sobel magnitude criterion is depicted. An alignment plane 80 is fitted to the nine phase points, by finding a least squares solution. The direction of the normal vector (nx, ny, nz) of the alignment plane 80 in reference to the z direction indicates the current orientation of the alignment plane. From this normal vector, values for pitch and yaw correction can be derived.Yaw and pitch may for example be obtained from the normal vector (nx, ny, nz) according to the relations tan(yaw) = nx / (-ny) and tan(pitch) = sqrt(nxA2 + nyA2) / nz .The correction values for pitch and yaw obtained in this way are finally displayed to the user who then can manually adjust the orientation of, e.g. the object plane, according to the principles set forth in Figs. 3, 4a, b and 5a, b above. Angle scales depicted on the pitch and yaw rotation platforms (see Figs. 4a, 5b) may help the user to dial in the correct pitch and yaw corrections.Fig. 9 shows a flow diagram of the multi-point refocusing process to realize object plane- and / or sensor plane-calibration in a digital in-line holographic microscope (DIHM). At S91, an image of a calibration target is captured with a digital in-line holographic microscope (DIHM). At S92, n- point focus distances of the phase points are obtained according to the Sobel magnitude criterion. At S93, the alignment plane is determined by least square optimization from distances and positions of the phase points. At S94, pitch and yaw corrections are determined based on the orientation of the alignment plane. At S95, the pitch and yaw corrections are presented to user.It should be noted with regard to S91, that determination of the image data as described above can also be performed with less than three different wavelengths. In particular, calibration with only one wavelength may be suitable here. That is, it is sufficient to calibrate the system using a single wavelength, and calibration is valid for all other wavelengths.Fig. 10 schematically shows exemplary components of a digital in-line holographic microscope (DIHM) configured to implement a multi-point refocusing process to realize object plane- and / or sensor plane-calibration. The in-line laser 8 is configured to illuminate the phase calibration target 60 with partially coherent time sequential illumination light beams. The image sensor 14 is configured to capture respective images of the calibration target 60. The processor 90 is configured to determine a quantitative phase image (QPI) and an amplitude image of the calibration target 60 based on the captured images. The processor 90 is further configured to execute the process described in Fig. 9 above. The processor 90 is configured to determine, from the image (e.g. QPI image, amplitude image, or the complex field information as such) of thecalibration target 60 and according to the Sobel magnitude criterion, the n-point focus distances of the phase points contained in the calibration target 60. The processor 90 is configured to determine the alignment plane by least square optimization from the distances and positions of the phase points. The processor 90 is further configured to determine pitch and yaw corrections based on the orientation of the alignment plane. The user interface 91 is configured to present these pitch and yaw corrections to the user, e.g., via a display screen of the digital in-line holographic microscope (DIHM). The pitch control 34 and yaw control 32 can be used by the user to achieve better alignment of the alignment plane.Fig. I la shows an exemplary user interface element generated by a user interface (91 in Fig. 10). The user interface element is presented to the user to indicate the result of the calibration process described in Fig. 9. In this example the result of the calibration process indicates that the yaw angle shows a misalignment of -1.2° and that the pitch angle shows a misalignment of -0.9°. The user interface element further indicates to the user that this misalignment is considered as “bad”. This should motivate the user to use the yaw control and the pitch control to achieve better alignment of the alignment plane.Fig. 1 lb shows another exemplary user interface element generated by a user interface (91 in Fig. 10). In this example the result of the calibration process indicates that the yaw angle shows a misalignment of +0.4° and that the pitch angle shows a misalignment of -0.3°. The user interface element further indicates to the user that this misalignment is considered as “fair”. This should indicate to the user that the alignment is good enough to achieve reasonable results, but that alignment could be better.Fig. 11c shows another exemplary user interface element generated by a user interface (91 in Fig. 10). In this example the result of the calibration process indicates that the yaw angle shows a misalignment of +0.3° and that the pitch angle shows a misalignment of -0.2°. The user interface element further indicates to the user that this misalignment is considered as “good”. This should indicate to the user that the alignment is good, but that alignment could be even better.Fig. l id shows another exemplary user interface element generated by a user interface (91 in Fig. 10). In this example the result of the calibration process indicates that the yaw angle shows a misalignment of +0.1° and that the pitch angle shows a misalignment of -0.2°. The user interface element further indicates to the user that this misalignment is considered as “excellent”. This should indicate to the user that the alignment is very good and that further efforts are unreasonable.The calibration process described above may improve quality and accuracy of the phase and amplitude reconstruction over the complete acquisition stack across the entire field-of-view (FoV), in particular for multi -wavelength systems.Calibration with polystyrene beadsIn another embodiment, the calibration target is a special calibration target using polystyrene (PS) beads as refocusing targets.Figs. 12a and 12b show (in a top-view and, respectively, a cross-sectional view) a microscope slide 95 surface-functionalized in a way that the cover glass contains covalently attached PS beads 96 (e.g., 5 pm diameter with carboxyl functional groups at their surface), such that all PS beads 96 form a monolayer structure 97, where they all reside in the same plane and the whole slide 95 is densely covered with PS beads 96. Thereby, the size of the beads 96, their size distribution (CV typ. 1-3%), and their density on the surface may be tailored to fit the respective requirements.Further, the PS bead target can be easily covered by a fluidic cell or brought into the fluidic cell, thus allowing tunability of the refractive index contrast between PS beads and their environment by applying a fluid of chosen refractive index as the host medium for the PS bead sample. This can be useful to avoid phase wrapping during the calibration process, in particular when calibrating the system at different wavelengths. Also, the refractive index fluid may be changed during the measurements and adapted to the respective wavelength under use and / or to increase data diversity.The specially tailored target for refocusing may be provided, e.g., as a separate item delivered with the main DIHM device. The PS bead target is used as component of the opto-mechanic part of the calibration process for adjustment of yaw or pitch in the object and / or sensor plane.Using this specially tailored PS bead target, calibration can be performed in a "one-shot"- approach, requiring only a single image to be acquired, since equally sized PS particles cover the entire FoV. This avoids that features of different dimension are spread across the target. Moving the target across the FoV, which would limit accuracy to the stage movement accuracy, is thus not necessary. Also, as the PS beads have a uniform size distribution, empirical correction of the size variation is not necessary. Further, the special PS bead targets are cost efficient so that a PS bead target may be provided with each produced DIHM for customer's convenience.AutomatizationIn the embodiments described above, the calibration is realized by hardware calibration that is manually controlled by the user. In the embodiment described below, this process is automated.Fig. 13 shows a computer-implemented process of automated pitch and yaw correction. At 84, a pitch correction 88 and a yaw correction 89 are determined based on the orientation of the alignment plane as described in Fig. 9 above in more detail. Pitch correction 88 is provided to pitch control 34. Pitch control 34 comprises mechanical means to automatically translate the received pitch correction 88 into an appropriate reconfiguration of the alignment plane. This may for example be achieved with a stepper motor. Yaw correction 89 is provided to yaw control 32. Yaw control 32 comprises mechanical means to automatically translate the received yaw correction 89 into an appropriate reconfiguration of the alignment plane. This may for example be achieved with a stepper motor. In this way, the calibration is automated.Computer-implementation of the processesFig. 14 schematically describes an embodiment of an electronic device which may implement the functionality of the process steps described above. The electronic device 1500 comprises a CPU 1501 as processor. The electronic device 1500 further comprises a GPU 1506 that is connected to the processor 1501. The electronic system 1500 further comprises an Ethernet interface 1504 which acts as interface for data communication with external devices, as for example a DH4M scanner. The DHIM scanner can also be connected to the electronic device with other standard connection buses, like USB. The electronic device 1500 further comprises a user interface 1505 that may present, e.g., via a display screen, pitch and yaw corrections to the user. The electronic device 1500 further comprises a data storage 1502 and a data memory 1503 (here a RAM). The data memory 1503 is arranged to temporarily store or cache data or computer instructions for processing by the processor 1501. The data storage 1502 is arranged as a longterm storage, e.g., for recording a scanned hologram or the labelled (in the sense of labelled for supervised learning) data which is necessary for the supervised learning of the classification algorithm. The data storage 1502 and the data memory 1503 may comprise computing instructions that implement the processes described above, e.g., a process of recording and storing a scanned hologram of an object. The computing instructions may further implement a process of decoding the hologram of the object to obtain phase and amplitude information of the object. The computing instructions may further implement the functionality of a calibration process for adjustment of yaw or pitch in the object and / or sensor plane as described in Fig. 9. The computing instructions may further implement a classification process, a process of training a classification algorithm as described in Fig. 2.***In so far as the embodiments of the disclosure described above are implemented, at least in part, using software-controlled data processing apparatus, it will be appreciated that a computer program providing such software control and a transmission, storage or other medium by which such a computer program is provided are envisaged as aspects of the present disclosure.Note that the present technology can also be configured as described below:[1] A device, the device being configured to determine multi-point refocusing information using a target (60, 95) to realize object plane (13)- and / or sensor plane (3 l)-calibration in a holographic microscope (1).[2] The device of [1], wherein the device is configured to provide information about an orientation of an alignment plane (80).[3] The device of [2], wherein the device is further configured to adjust the orientation of the alignment plane (80).[4] The device of [3], wherein the device is configured to provide for pitch and yaw correction.[5] The device of [4], the device comprising a pitch rotation platform (33) and a yaw rotation platform (35) to provide pitch and yaw correction.[6] The device of [1], wherein the device is configured to provide n-point refocusing information obtained from a phase image of a phase target (60).[7] The device of [6], wherein the device is configured to apply the Sobel magnitude criterion for obtaining the n-point refocusing information.[8] The device of [1], comprising a calibration target with multiple refocusable structures, where the locations of the refocusable structures span a 2D plane in 3D space.[9] The device of [8], wherein the calibration target is a phase calibration target.

[0010] The device of [8], wherein the calibration target uses polystyrene beads (96) as refocusing targets.

[0011] The device of [8], wherein the calibration target comprises a slide (95) surface- functionalized in a way that the cover glass contains covalently attached polystyrene beads (96).

[0012] The device of

[0010] , wherein the polystyrene beads (96) have a diameter of between 2 and 10 pm.

[0013] The device of

[0010] , wherein the polystyrene beads (96) are provided with carboxyl functional groups at their surface.

[0014] The device of

[0010] , wherein the polystyrene beads (96) are configured to form a monolayer structure (97).

[0015] The device of

[0011] , wherein the slide (95) is densely covered with polystyrene beads (96).

[0016] The device of anyone of

[0010] to

[0015] , wherein the polystyrene beads (96) are of substantially equal size.

[0017] The device of anyone of

[0010] to

[0016] , wherein the size distribution of the polystyrene beads (96) is such that the coefficient of variation is smaller than 3%.

[0018] A method comprising determining multi-point refocusing information using a target (60, 95) to realize object plane (13)- and / or sensor plane (31)-calibration in a holographic microscope (1).

[0019] The method of

[0018] , wherein the method comprises capturing (S91) an image of a calibration target with a holographic microscope (1).

[0020] The method of

[0018] or

[0019] , wherein the method further comprises obtaining (S92) n- point focus distances of phase points according to the Sobel magnitude criterion.

[0021] The method of anyone of

[0018] to

[0020] , wherein the method further comprises determining(593) an alignment plane (80) from distances and positions of the phase points.

[0022] The method of anyone of

[0018] to

[0021] , wherein the method further comprises determining(594) pitch and yaw corrections based on an orientation of the alignment plane (80).

[0023] The method of anyone of

[0018] to

[0022] , wherein the method further comprises presenting(595) pitch and yaw corrections to the user.

[0024] The method of anyone of

[0018] to

[0023] , wherein the method comprises automatically controlling pitch and yaw according to a determined pitch correction (88) and a determined yaw correction (89).

[0025] The method of anyone of

[0018] to

[0024] , wherein the method comprises covering a polystyrene bead target (95) by a fluidic cell or bringing it into the fluidic cell comprising a fluid with a given refractive index.

[0026] The method of

[0025] , comprising changing the fluid during measurements to adapt it to the respective wavelength under use.

[0027] The method of

[0025] , comprising changing the fluid during measurements to increase data diversity.

[0028] A phase calibration target (60) for use in a multi-point refocusing process to realize object plane- and / or sensor plane-calibration in a holographic microscope (1).

[0029] The phase calibration target (60) of

[0028] , comprising multiple refocusable structures, where the locations of the refocusable structures span a 2D plane in 3D space.

[0030] A calibration target, wherein the calibration target comprises polystyrene beads (96) as refocusing targets.

[0031] The calibration target of

[0030] , wherein the polystyrene beads (96) span a 2D plane (97) in 3D space.

[0032] The calibration target of anyone of

[0030] to

[0031] , wherein the calibration target comprises a slide (95) surface-functionalized in a way that the cover glass contains covalently attached polystyrene beads (96).

[0033] The calibration target of anyone of

[0030] to

[0032] , wherein the polystyrene beads (96) have a diameter of 5 pm.

[0034] The calibration target of anyone of

[0030] to

[0033] , wherein the polystyrene beads (96) are provided with carboxyl functional groups at their surface.

[0035] The calibration target of anyone of

[0030] to

[0034] , wherein the polystyrene beads (96) are configured to form a monolayer structure (97).

[0036] The calibration target of anyone of

[0032] to

[0035] , wherein the slide (95) is densely covered with polystyrene beads (96).

[0037] The calibration target of anyone of

[0030] to

[0036] , wherein the polystyrene beads (96) are of substantially equal size.

[0038] The calibration target of anyone of

[0030] to

[0037] , wherein the size distribution of the polystyrene beads (96) is such that the coefficient of variation is smaller than 3%.Reference signs1 digital in-line holographic microscope6 illumination light beams8 in-line holography laser12 object13 object plane14 image sensor30 multi-hight acquisition31 sensor plane32, 34 rotary control33, 35 rotation platform37 scale38 screw60 calibration target80 alignment plane90 processor91 user interface95 microscope slide96 polystyrene (PS) beads97 monolayer structure1500 electronic device1501 processor1502 data storage1503 data memory1504 Ethernet interface1505 user interface1506 GPU

Claims

CLAIMS1. A device, the device being configured to determine multi-point refocusing information using a target to realize object plane- and / or sensor plane-calibration in a holographic microscope.

2. The device of claim 1, wherein the device is configured to provide information about an orientation of an alignment plane.

3. The device of claim 2, wherein the device is further configured to adjust the orientation of the alignment plane.

4. The device of claim 3, wherein the device is configured to provide for pitch and yaw correction.

5. The device of claim 4, the device comprising a pitch rotation platform and a yaw rotation platform to provide pitch and yaw correction.

6. The device of claim 1, wherein the device is configured to provide n-point refocusing information obtained from a phase image of a phase target.

7. The device of claim 6, wherein the device is configured to apply the Sobel magnitude criterion for obtaining the n-point refocusing information.

8. The device of claim 1, comprising a calibration target with multiple refocusable structures, where the locations of the refocusable structures span a 2D plane in 3D space.

9. The device of claim 8, wherein the calibration target is a phase calibration target.

10. The device of claim 8, wherein the calibration target uses polystyrene beads as refocusing targets.

11. The device of claim 8, wherein the calibration target comprises a slide surface- functionalized in a way that the cover glass contains covalently attached polystyrene beads.

12. The device of claim 10, wherein the polystyrene beads have a diameter of between 2 and 10 pm.

13. The device of claim 10, wherein the polystyrene beads are provided with carboxyl functional groups at their surface.

14. The device of claim 10, wherein the polystyrene beads are configured to form a monolayer structure.

15. The device of claim 11, wherein the slide is densely covered with polystyrene beads.

16. The device of claim 10, wherein the polystyrene beads are of substantially equal size.

17. The device of claim 10, wherein the size distribution of the polystyrene beads is such that the coefficient of variation is smaller than 3%.

18. A method comprising determining multi-point refocusing information using a target to realize object plane- and / or sensor plane-calibration in a holographic microscope.

19. The method of claim 18, wherein the method comprises capturing an image of a calibration target with a holographic microscope.

20. The method of claim 18, wherein the method further comprises obtaining n-point focus distances of phase points according to the Sobel magnitude criterion.

21. The method of claim 18, wherein the method further comprises determining an alignment plane from distances and positions of the phase points.

22. The method of claim 18, wherein the method further comprises determining pitch and yaw corrections based on an orientation of the alignment plane.

23. The method of claim 18, wherein the method further comprises presenting pitch and yaw corrections to the user.

24. The method of claim 18, wherein the method comprises automatically controlling pitch and yaw according to a determined pitch correction and a determined yaw correction.

25. The method of claim 18, wherein the method comprises covering a polystyrene bead target by a fluidic cell or bringing it into the fluidic cell comprising a fluid with a given refractive index.

26. The method of claim 25, comprising changing the fluid during measurements to adapt it to the respective wavelength under use.

27. The method of claim 25, comprising changing the fluid during measurements to increase data diversity.

28. A phase calibration target for use in a multi-point refocusing process to realize object plane- and / or sensor plane-calibration in a holographic microscope.

29. The phase calibration target of claim 28, comprising multiple refocusable structures, where the locations of the refocusable structures span a 2D plane in 3D space.

30. A calibration target, wherein the calibration target comprises polystyrene beads as refocusing targets.

31. The calibration target of claim 30, wherein the polystyrene beads span a 2D plane in 3D space.

32. The calibration target of claim 30, wherein the calibration target comprises a slide surface-functionalized in a way that the cover glass contains covalently attached polystyrene beads.

33. The calibration target of claim 30, wherein the polystyrene beads have a diameter of 5 pm.

34. The calibration target of claim 30, wherein the polystyrene beads are provided with carboxyl functional groups at their surface.

35. The calibration target of claim 30, wherein the polystyrene beads are configured to form a monolayer structure.

36. The calibration target of claim 32, wherein the slide is densely covered with polystyrene beads.

37. The calibration target of claim 30, wherein the polystyrene beads are of substantially equal size.

38. The calibration target of claim 30, wherein the size distribution of the polystyrene beads is such that the coefficient of variation is smaller than 3%.

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