Light source array, imaging system for Fourier stack-up imaging, and method for performing Fourier stack-up imaging

Abstract

The present disclosure relates to an array of light sources (2) for an imaging system (100) for Fourier stack-up imaging, comprising at least two adjacent light sources (2), preferably different from each other in terms of characteristics of the light beams emitted from them, and configured to illuminate a sample (4) at different angles of incidence, and a plurality of light sources (2) for reconstructing a single image of the sample using Fourier stack-up imaging wherein the characteristics of the light beam of each light source (2) are configured to match the sample (4) to increase contrast and / or color or material information content in the reconstructed single image of the sample (4). In embodiments, the one or more light sources are UV light sources. In an embodiment, the sample is a biological sample. In an embodiment, the FPM includes a UV transmissive component and / or a material such as quartz.

Description

Technical Field

[0001] The present disclosure relates to light source arrays (such as ultraviolet light or "UV") for imaging systems for Fourier stylus imaging, the imaging systems for Fourier stylus imaging preferably including one or more UV light sources, and methods of performing Fourier stylus imaging preferably using an ultraviolet (UV) based system for unstained imaging of biological specimens. Background Art

[0002] Fourier stacking microscopy (FPM) is a computational microscopy technique that generates high-resolution images over a large field of view (FoV). This is achieved by acquiring a sequence of images of a region of interest of a specimen in brightfield mode and / or darkfield mode at various illumination angles greater than the numerical aperture (NA) of the objective used, and iteratively combining these images into a single high-resolution image in Fourier space.

[0003] The images may be taken by a FPM microscope, which may be assumed to be optically corrected to take images of the specimen with reasonably good quality without significant distortion or chromatic aberration.

[0004] Typically, Fourier ptychography images are acquired with a monochrome camera and color illumination, for example, by white light or a light source with a broader spectrum plus a bandpass filter with a selected wavelength or preferably a light emitting diode (LED) of narrow bandwidth. As illuminator or light source, a planar array or a hemispherical arrangement of LEDs can be used. Some systems also have narrow bandwidth bandpass filters to even further reduce the bandwidth of the light in a particular image. The illumination wavelength can be selected by an LED used in front of a monochrome camera or in combination with a bandpass filter with a full width at half maximum (FWHM) of, for example, 10, 50 or 80 nm. A color image consists of a set of separate images taken at different wavelengths, which are then individually reconstructed into a (quasi) monochrome high-resolution image. If desired, these high-resolution images can be combined afterwards to form a color image.

[0005] Microscopic samples, such as biological samples and medical samples, usually include different materials or tissues, but there is also a problem of relatively low or quite low material contrast at a given or specific wavelength for different materials in the corresponding sample area. Therefore, in the final reconstructed image using Fourier stylus imaging, changes in the material or changes in the material composition of the sample cannot be detected or cannot be clearly distinguished.

[0006] Therefore, there is a continuing need to have images with improved contrast and / or to provide solutions for Fourier ptychography that increase and optimize material differentiation in a single reconstructed image of a sample. Summary of the invention

[0007] This object is achieved inventively by the subject matter of the independent claim. Advantageous further developments can be derived from the dependent claims.

[0008] In an embodiment, according to a first aspect, the present disclosure provides a light source array for a system for Fourier staggered imaging, comprising at least two adjacent light sources, the at least two adjacent light sources being different from each other in the characteristics of the light beams emitted by them, and being configured to illuminate a sample at different incident angles to reconstruct a single image of the sample using Fourier staggered imaging, wherein the characteristics of the light beam of each light source are configured to match the sample to increase the contrast and / or color or material information content in the reconstructed single image of the sample. In an embodiment, one or more light sources are predetermined to provide UV light.

[0009] In an embodiment, the present disclosure utilizes the differences in spectral content of the characteristics of light beams from different light sources to improve and optimize the material contrast of the entire image set, i.e., between different sub-images when capturing images of the image set, so as to better detect and distinguish changes in the material or material composition of the sample.

[0010] With the light source array of the present invention, the number of sub-images of a single image set does not need to be increased. Instead, the signal of a single image for a given illumination direction or overlapping multiple directions is optimized by increasing the absorption contrast, or by optimizing the diffraction efficiency or sensitivity to refraction changes, to increase and optimize material discrimination.

[0011] In embodiments of the light source array, the characteristics of the light beam include the wavelength, bandwidth, spectral curve and / or polarization of the light beam.

[0012] In an embodiment, the wavelength of the light beam emitted from the light source may range from UV (including DUV < 200 nm) through VIS to IR (including SWIR and NIR up to 2500 nm). The wavelength of the light beam is limited only by the camera technology and the optical system to produce a suitable image.

[0013] In embodiments, it may be advantageous to mix light sources that differ from each other in terms of wavelength bandwidth / spectrum and / or polarization of the generated light beams for a single reconstruction.

[0014] Approaches using, for example, multi-wavelength light sources enable images to be generated with optimized or customized contrast settings even for samples that are difficult to image at a single given wavelength.

[0015] Furthermore, in some embodiments, filters can be used that can shape the intensity distribution of an LED into a desired and quite arbitrary shape, such as multiple bands from a single LED.

[0016] In a further embodiment of the array of light sources, the contrast in the reconstructed single image of the sample depends on the interaction characteristics of the light beams of the respective light sources with a given position of the sample.

[0017] Embodiments of the present disclosure may exploit the fact that a single image captured with, for example, different wavelengths may have the same physical locations and features on the image sensor, but they may differ in contrast based on the respective interaction characteristics of the respective wavelengths of light with specific locations of the sample.

[0018] In another preferred embodiment of the array of light sources, the interaction properties comprise absorption, refraction, scattering and / or diffraction of the light beams of the respective light sources at a given position of the sample.

[0019] In another preferred embodiment of the light source array, the interaction characteristics depend on the properties of the light beams of the respective light sources and on the material and / or geometry of the sample at a given location.

[0020] In another preferred embodiment of the light source array, the light source array includes a plurality of, for example at least three, light sources, which are a first light source, a second light source and a third light source, wherein the first light source is adjacent to the second light source, and the second light source is further adjacent to the third light source, wherein the first light source and the second light source are different from each other in characteristics of light beams emitted by them, wherein the second light source and the third light source are different from each other in characteristics of light beams emitted by them, and wherein the first light and the third light source include the same characteristics from the light beams emitted by them.

[0021] In an FPM system, adjacent light sources in the light source array should have at least 35% overlap in aperture or Fourier space. The overlap is required to properly stitch image content from different illumination angles.

[0022] In an embodiment, adjacent light sources may have different wavelengths. However, a further light source - a third light source, such as the next light source, has the same wavelength as the first light source. In an embodiment, one or more light sources may include one or more wavelengths characterized as ultraviolet.

[0023] The second light source, acting as an "intermediate" light source, is used to ensure that the image reconstruction algorithm properly stitches two adjacent light sources of the same type to make contributions that are properly aligned in Fourier space.

[0024] This assignment of light sources with different wavelengths is due to the fact that there is a physical reconstruction directly from the Fourier transform of the color-corrected image, while omitting a further transformation to an angular coordinate system usually used in Fourier ptychography.

[0025] In another preferred embodiment of the light source array, in a reconstruction process using Fourier phantom imaging, images recorded with the aid of the second light source include lower weighting factors than images recorded with the aid of the first and third light sources.

[0026] When homogenizing information in an iterative reconstruction cycle, the information of the "intermediate" light sources is fitted or used with smaller weighting factors, or only with moderate weighting factors, to appropriately stitch together the information of light sources of the same characteristics, such as wavelength, bandwidth or polarization.

[0027] This approach can be similar to the same kind of reconstruction for monochrome images in a typical reconstruction. However, the number of sub-images for different wavelengths is significantly reduced.

[0028] This method of alternating light sources of different wavelengths can be used for a small number of wavelengths, such as 2 or 3 different wavelengths, which are preferably arranged in a rectangular hexagonal or circular shape in a planar or 3D form with equal or linear frequency modulation distances from the center to the high aperture light source.

[0029] In another preferred embodiment of the light source array, during reconstruction using Fourier phantom imaging, the image captured with the aid of the second light source comprises the same weighting factors as the images captured with the aid of the first and third light sources.

[0030] Thereby, the "middle" light source is weighted the same as the other light sources, regardless of the respective color. In this case, the respective information content of all images contributes to the final composite image with the same weight, regardless of the wavelength used.

[0031] In this way, a common resolution can be achieved where different wavelengths contribute to the final image. This is independent of whether it is an image taken by a monochrome sensor, a color image taken by a color sensor, or an image generated from multiple monochrome images with different illumination settings.

[0032] In another preferred embodiment of the light source array, the light source array comprises a plurality of light sources, in particular more than three light sources, preferably more than five light sources, wherein the plurality of light sources form at least two clusters, wherein the light sources within each cluster are adjacent to each other, and wherein the characteristics of the light beams of the light sources within each cluster are configured to match a given position of the sample so as to increase the contrast in a reconstructed single image of the given position of the sample.

[0033] Thereby, light sources of different spectral content (e.g. wavelength, bandwidth and polarization) are arranged as quasi (local) clusters for each type of light source. For example, the central light source used for the bright field imaging part is more used for absorption contrast and is therefore preferably selected according to the sample characteristics, while the dark field light source is more used for the angular scattering properties of the sample with respect to refraction, dispersion and / or diffraction, where absorption may be hindered because the light path in the sample is extended and thus the signal is more attenuated along its propagation path.

[0034] In another preferred embodiment of the light source array, the light source array is configured to be selected based on the type of sample.

[0035] For example, for unstained biological samples, such as body fluids, blood cells, blood smears, tissues and tissue sections, the absorption contrast in the visible spectrum is quite weak. Therefore, the bright field imaging light source can be selected from the non-visible spectral range such as UV or IR. In the UV range, there are different absorption mechanisms based on the carbon bonds of organic compounds, which can produce quite strong absorption contrast in the range of 200-380nm, especially in the range of 250-320nm, which can be easily achieved by using commercial LEDs. Preferably, the light source is configured to emit light in the range of 200 to 380nm, most preferably in the range of 250 to 320nm.

[0036] In an embodiment, according to a second aspect, the present disclosure provides an imaging system for Fourier stack imaging, which comprises a light source array according to the first aspect.

[0037] In an embodiment of the imaging system of Fourier stack imaging, the imaging system of Fourier stack imaging includes a bandpass filter, a notch filter, a filter defining a complex spectral curve and / or an edge filter arranged between the light source array of the imaging system of Fourier stack imaging and the camera sensor. The filter is preferably inserted into the imaging beam path between the objective lens and the camera.

[0038] Thereby, spectral characteristics and / or material differences in (absorption) contrast are improved. Dark field illumination can consist of different, e.g. longer wavelengths, e.g. in the VIS or IR range, since beams in these ranges can be scattered more efficiently depending on the characteristic size of the sample being measured.

[0039] In another preferred embodiment of the imaging system for Fourier stack imaging, the imaging system for Fourier stack imaging comprises an objective lens, wherein the objective lens aperture of the objective lens can be divided into a plurality of parts, wherein each part of the objective lens aperture is associated with a light source or a light source cluster in the light source array.

[0040] In order to further exploit the local clustering of light sources, the illumination system can be constructed in such a way that the entire objective aperture range or the objective aperture in the brightfield and / or darkfield illumination portions is divided into a plurality of segments / areas, for example two, three, four, six or eight segments, and each segment is equipped with a different light source or a different combination of light sources (in terms of wavelength, bandwidth and / or polarization) used in the illumination system.

[0041] As described above, in a preferred embodiment of the light source array, each light source may have nearest neighbors of a different type. However, light sources of the same type are preferably arranged in such a way that a larger segment / portion of the illumination and / or imaging aperture space is filled for that particular light source type or a given combination of light source types.

[0042] Due to the high symmetry in Fourier space, sectors at point-symmetric positions can use completely different types of light sources to optimize the sampling efficiency of samples during imaging and reconstruction. For example, when the light source has four, six or eight segments, there may be two, two or three, or two or four different light source selections for the corresponding segments. These combinations can be selected for adjacent segments in a given sequence. For example, in the case of six segments and two types of light sources, each type has three segments, and adjacent segments have different types of light sources. This leads to a setting in which no segment is opposite to another segment of the same type, showing a wider coverage range in aperture space for one type / a group of wavelengths, and even for small details of the object, no information is lost. If the adjacent light sources in the radial direction also include different types to optimize the sampling strategy, the sampling of Fourier space can be further optimized for the sampling uniformity of a given light source type.

[0043] According to a third aspect, the present invention provides a method for performing Fourier stacking imaging, comprising: illuminating a sample at different incident angles by at least two adjacent light sources in a light source array, wherein the at least two adjacent light sources differ from each other in the characteristics of the light beams emitted by them, and the characteristics of the light beam of each of the at least two light sources match the sample to increase the contrast in a single image of the sample to be reconstructed; acquiring an image of the sample by a camera with the aid of the light beams from the at least two adjacent light sources; and reconstructing a single image of the sample by using the captured image.

[0044] In a preferred embodiment of the method, the characteristics of the light beam include the wavelength, bandwidth and / or polarization of the light beam. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The present invention will be explained in more detail with reference to the exemplary embodiments depicted in the attached drawings. The accompanying drawings are intended to provide a further understanding of the present invention and are incorporated into and constitute a part of this specification. The accompanying drawings illustrate embodiments of the present invention and together with the description serve to explain the principles of the present invention.

[0046] Other embodiments of the present invention and many of the intended advantages of the present invention will be readily understood as they become better understood by reference to the following detailed description. Like reference numerals represent corresponding similar parts. The accompanying drawings are not drawn to scale.

[0047] Figure 1 a shows in perspective the arrangement of an imaging system for Fourier stack imaging including a light source array according to an embodiment of the present invention;

[0048] Figure 1 b shows the Figure 1 Fourier spectrum of a sample disposed at the rear focal plane of the objective lens in the imaging system of Fourier stack imaging shown in a;

[0049] Figure 1 c shows the Figure 1 The imaging system of Fourier stack imaging shown in a is set up to change the illumination angle by illuminating the LED at different positions;

[0050] Figure 2 shows an illumination device including a light source array according to another embodiment of the present invention;

[0051] Figure 3 shows a view of a light source array in a sample plane according to another embodiment of the present invention;

[0052] Figure 4 shows a view of a light source array in a sample plane according to another embodiment of the present invention;

[0053] Figure 5 A block diagram illustrating an embodiment of a method of performing Fourier phantomography;

[0054] Figure 6 depicts a graph with data relating to UV absorption of proteins and nucleic acids;

[0055] Fig. 7A and 7b Depicted are images of an unstained blood smear on a quartz slide imaged once at (A) 600 nm and (b) 280 nm; and

[0056] Figure 8Depicted is an FPM reconstruction of an image of a blood smear imaged at 280 nm. The left image shows a low-resolution image acquired by the center LED before reconstruction. The image on the right corresponds to a high-resolution image generated by combining the image on the left with all other images acquired at different illumination angles in Fourier space. DETAILED DESCRIPTION

[0057] Figure 1 FIG. 1 shows a perspective view of an imaging system 100 for Fourier stack imaging according to an embodiment of the present invention, the system comprising an array of light sources 2, the light sources 2 being placed on a carrier 3. The imaging system 100 for Fourier stack imaging further comprises a sample 4 placed in a sample plane 5 from bottom to top, an objective lens 6, a tube lens 10, and a camera 12 for collecting an image of the sample 4. Figure 1 As shown in FIG. 1 , the region of interest 7 of the sample 4 is illuminated by the array of light sources 2 that flash continuously to achieve illumination at a specific illumination angle. The hollow rectangle 8 represents the back focal plane of the objective lens 6 and / or the aperture plane corresponding to the objective lens 6.

[0058] exist Figure 1 In the embodiment shown in a, the light source 2 array includes a spatial distribution of separate small aperture LEDs, the number of which is 15*15=225. The exact number and arrangement of LEDs is determined by the desired resolution of the reconstructed image. In other embodiments not shown, laser-based or fiber-based light sources are possible.

[0059] like Figure 1 As shown in a, LED2 is positioned on the plane in a grid shape with equal spacing in the x and y directions. In other embodiments not shown, LEDs are positioned on the plane in a grid shape with equal spacing only in the x direction or only in the y direction. In another embodiment, LED2 is positioned on the plane in a grid shape, and adjacent light sources have a linear frequency modulation interval so that the effective angular spacing seen from the object is regular, that is, equidistant in shape.

[0060] In another embodiment, the light sources 2 are arranged on a three-dimensional structure such as a hemisphere, where each light source has the same effective geometric distance from the center of the field of view of the optical device, and in a preferred embodiment, the optical axis of each LED is pointed to the center of the field of view of the optical system in the best focus plane setting. In general, any other spatial distribution of effective light sources is possible. A specific design can be calibrated to establish appropriate image reconstruction performance of the FPM system.

[0061] In some embodiments, monochromatic or polychromatic light sources are mounted at specific locations of the grid and are preferably individually controlled in their intensity over time, e.g. switched on or off, and are set with appropriate power levels which may be fixed pre-calibrated and preferably linearized for adaptive settings.

[0062] Traditionally, all grid positions are equipped with the same type of light source. This design is essentially dictated by the requirements of the reconstruction algorithm for (quasi-)monochromatic image sets. However, not all wavelengths are equally useful in all possible positions of the illumination grid. For example, it is conceivable that a wavelength matching a strong absorption band of the specimen might be preferred for illuminating the brightfield region of the system, while a darkfield light source matches a wavelength region where the scattering cross section of the specimen is large.

[0063] In an embodiment, different light sources having, for example, different but known wavelengths and / or bandwidths are arranged at different positions of the grid, and there is no need to have nominally identical light sources at all grid positions.

[0064] Single images taken with, for example, different wavelengths have the same physical location on the sample and, therefore, feature structures on the image sensor of the camera 12, but they may differ in contrast depending on the respective interaction properties of the light of the respective wavelength with the specific location of the sample 4. The interaction properties include absorption, refraction, scattering, diffraction and / or fluorescence properties of the light beam of the respective light source 2 at a given location 7 of the sample 4. The interaction properties depend on the properties of the light beam of the respective light source, such as the wavelength, bandwidth and / or polarization of the light beam, and also on the material and / or geometry at the given location 7 of the sample 4.

[0065] By means of an array of light sources 2, the number of sub-images of a single image set does not need to be increased. Instead, material differentiation is increased and optimized by, for example, increasing the absorption contrast or by optimizing the diffraction efficiency or sensitivity to refraction changes, optimizing the signal of a single image for a given illumination direction or overlapping multiple directions.

[0066] Figure 1 b shows the Figure 1 The Fourier spectrum of the sample 4 arranged at the back focal plane of the objective lens in the imaging system of Fourier stack imaging shown in a. The circular sub-area 20 corresponds to the aperture size of the objective lens 6 and moves with the movement of the illumination angle.

[0067] The light field emitted from one LED 2 can be approximated as a plane wave in a small region at the sample plane, because the large distance between LED 2 and sample 4 increases the spatial coherence of LED 2. The plane wave has vector components (kx, ky) associated with the oblique illumination of LED 2 on sample 4. Lighting up one LED 2 causes the Fourier spectrum of the sample to shift (kx, ky) at the back focal plane of the objective, and the finite numerical aperture (NA) of the objective acts as a low-pass filter that transmits only a small sub-region at the center of the shifted Fourier spectrum. The low-pass Fourier component propagates further to the image plane and is acquired by camera 12. LEDs 2 are sequentially illuminated so that the acquired image contains partially overlapping Fourier sub-regions that together span the entire Fourier spectrum of sample 4.

[0068] exist Figure 1 In c, a part of the LED array on the carrier board 3 is shown in an enlarged view. Figure 1 The parallel arrows and the parallel dashed lines shown in c represent the sequential switching of LEDs 2 at different positions on the matrix to achieve the movement of the illumination angle. Figure 1 In the state shown in c, LED2 (I) is turned on to provide illumination.

[0069] Figure 2 An illumination device 14 is shown, comprising a base unit 16 and a hemispherical carrier 18 fixed to the base unit 16. A hemispherical arrangement of LEDs (not shown) is provided on the hemispherical carrier 18, wherein each LED has the same effective geometric distance from the center of the field of view of the optical device. Due to the hemispherical arrangement, the optical axis of each LED points to the symmetry axis of the hemisphere, and all optical axes should intersect at the nominal focus position of the optical device to improve the illumination uniformity of the field of view.

[0070] Figure 3 A view of a light source array according to another embodiment of the present invention in a sample plane 5 is shown. In this embodiment, 9*9=81 LEDs 2 arranged in a planar form are used to form a rectangular illuminator.

[0071] Figure 4 FIG. 5 shows a view of a light source array according to another embodiment of the present invention in a sample plane 5. In this embodiment, a total of 76 LEDs 2 arranged in three dimensions are used to form a hemispherical illuminator. The LEDs 2 are at a constant radial distance from the intersection of the optical axis (+) and the sample plane 5. Figure 4 In the illustrated view, the angular increments of the rings formed by the LEDs are fixed, and for each ring the LEDs have a "constant" angular spacing. An individual azimuth offset can be set for each ring to avoid overtone effects.

[0072] Figure 5The illustrated embodiment of the method for performing Fourier stacking includes three steps. In step S10, the sample is illuminated at different angles of incidence by at least two adjacent light sources in a light source array, wherein the two adjacent light sources differ from each other in the characteristics of the light beams emitted by them, and the characteristics of the light beams of each of the at least two light sources match the sample to increase the contrast in a single image of the sample to be reconstructed. In step S20, an image of the sample is captured by a camera with the aid of light beams from at least the two adjacent light sources. In step S30, a single image of the sample is reconstructed using the captured image. In an embodiment, additional steps are included, such as providing one or more biological samples, such as body fluids, blood cells, blood smears, tissues, and tissue sections. In an embodiment, one or more UV light waves are preselected for contact with the biological sample or specimen to increase the contrast of the resulting image.

[0073] The invention is not limited to microscopy but has general application to Fourier stylus imaging.

[0074] In some embodiments, (local) clustering of light sources with wavelengths can also be used to further extend the NA depending on the reconstruction method. When the wavelength becomes shorter for a higher NA of the illumination, or there is some overlap with the normal wavelength of the illumination for the corresponding image set, the effective maximum NA of the reconstructed image can be further increased, even if the reconstructed image is based on a different wavelength.

[0075] In embodiments, to optimize the image quality of the images captured for a single image set, it may be preferred to select a wavelength for illumination that corresponds to the design wavelength of the imaging optics for color corrections such as achromats and apochromats. If this is not possible, it is preferred to optimize the settings so that illuminations of the same focus setting are grouped into subgroups, and one of the subgroups is used after another. To further optimize the settings, the subgroups are preferably ordered so that changes from one subgroup to the next change the focus position in the same direction, for example in positive or negative increments. This allows minimizing movement and therefore reducing any effects from mechanical lag, or allows minimizing the execution time of the image acquisition process because the mechanical movement becomes minimized.

[0076] In an embodiment, by using the overlap of different wavelengths of image data in Fourier space based on color corrected images in the image plane, it is possible to use one complete image set / a given number of original images to generate separate synthetic images for one or more of the wavelengths used in data acquisition. Compared to standard reconstruction methods, an aspect of image synthesis is provided, which has reduced weight, or only appropriate stitching and alignment in the stitching of more distant image information of the same wavelength, or has greatly reduced overlap.

[0077] Image acquisition is based on different wavelengths that are employed to refocus the optics for the corresponding wavelength or bandwidth for optimal image contrast to compensate for chromatic (focus) aberrations of the optics. Refocusing may also mean rescaling the image, since when the focus position changes, the effective magnification may also change slightly. This dependence of magnification on focus position and / or wavelength can be determined in a standard calibration routine to determine the system magnification, for example by using a scale bar or length artifact or as a relative scale factor related to an appropriate test structure such as a binary pattern in a chrome-on-glass structure. With these calibration data, images acquired at a given wavelength can be recalibrated in size before their application in the reconstruction of Fourier stack imaging.

[0078] In some embodiments, the present disclosure relates to an array of light sources (2) of an imaging system (100) for Fourier staggered imaging, comprising at least two adjacent light sources (2), the at least two adjacent light sources differing from each other in the characteristics of the light beams emitted by them, and configured to illuminate a sample (4) at different incident angles to reconstruct a single image of the sample using Fourier staggered imaging. Wherein the characteristics of the light beam of each light source (2) are configured to match the sample (4) to increase the contrast and / or color or material information content in the reconstructed single image of the sample (4). In some embodiments, the characteristics of the light beams include the wavelength, bandwidth, spectral curve and / or polarization of the light beams. In an embodiment, the characteristics of the light beams are preselected to include light beams having wavelengths characteristic of being ultraviolet. In an embodiment, the contrast in the reconstructed single image of the sample (4) depends on the interaction characteristics of the light beams of the corresponding light sources (2) with a given position of the sample (4). In an embodiment, the interaction characteristics include absorption, refraction, scattering and / or diffraction of the light beams of the corresponding light sources (2) at a given position of the sample (4). In an embodiment, the interaction characteristics depend on the characteristics of the beams of the respective light sources (2) and the material and / or geometry at a given position of the sample (4). In an embodiment, the array of light sources (2) comprises at least three light sources (2), wherein a first light source is adjacent to a second light source, the second light source is further adjacent to a third light source, wherein the first light source and the second light source differ from each other in the characteristics of the beams emitted by them, wherein the second light source and the third light source differ from each other in the characteristics of the beams emitted by them, and wherein the first light source and the third light source comprise the same characteristics of the beams emitted by them. In an embodiment, during reconstruction using Fourier staging, an image captured with the aid of the second light source comprises a lower weighting factor than an image captured with the aid of the first and third light sources. In an embodiment, during reconstruction using Fourier staging, an image captured with the aid of the second light source comprises the same weighting factor as an image captured with the aid of the first and third light sources. In an embodiment, the array of light sources (2) comprises a plurality of light sources (2), in particular more than three light sources (2), preferably more than five light sources (2), wherein the plurality of light sources form at least two clusters, wherein the light sources within each cluster are adjacent to each other, and wherein the characteristics of the light beams of the light sources within each cluster are configured to match a given position of the sample (4) to increase the contrast in a reconstructed single image of the given position of the sample (4). In an embodiment, the array of light sources is configured to be selected based on the type of the sample (4).

[0079] In some embodiments, the present disclosure includes an imaging system (100) for Fourier stack imaging, which includes an array of light sources as shown or described herein. In an embodiment, the imaging system for Fourier stack imaging of the present disclosure includes a bandpass filter, a notch filter, a filter that limits a complex spectral curve, and / or an edge filter arranged between a camera sensor (12) of the imaging system (100) for Fourier stack imaging and the array of light sources (2). In an embodiment, the imaging system for Fourier stack imaging of the present disclosure includes an objective lens (6), wherein an objective lens aperture of the objective lens (6) can be divided into a plurality of segments, wherein each segment of the objective lens aperture is associated with a light source (2) or a cluster of light sources (2) in an array of light sources (2).

[0080] In an embodiment, an imaging system (100) for Fourier stack imaging includes one or more UV transmissive components, such as components including or consisting of quartz. Non-limiting examples of UV transmissive components include one or more of the following: a light source (2), a carrier plate (3), a sample plane (5), an objective lens (6), a region of interest (7), a hollow rectangle (8), a tube lens (10), a camera (12) or a UV transmissive component thereof, an irradiation device (14) or a UV transmissive component thereof.

[0081] In some embodiments, the present disclosure relates to a biological sample embedded in a refractive index matching medium. The refractive index matching medium is preferably UV transmissive, such as silicone oil.

[0082] In an embodiment, the present disclosure includes a method for performing Fourier staggered imaging, the method comprising: illuminating (S10) a sample at different incident angles by at least two adjacent light sources in an array of light sources, wherein the at least two adjacent light sources differ from each other in characteristics of light beams emitted by them, and the characteristics of the light beam of each of the at least two light sources match the sample for increasing the contrast in a single image of the sample to be reconstructed; acquiring (S20) an image of the sample by a camera with the aid of the light beams from the at least two adjacent light sources; and reconstructing (S30) a single image of the sample by using the captured image. In an embodiment, the characteristics of the light beam include a wavelength, a bandwidth and / or a polarization of the light beam. In an embodiment, illuminating (S10) the sample at different incident angles also includes illuminating with ultraviolet light. In an embodiment, illuminating (S10) the sample at different incident angles also includes providing one or more biological samples of the present disclosure.

[0083] In some embodiments, the present disclosure relates to an array of light sources (2) of an imaging system (100) for Fourier staggered imaging, comprising at least two adjacent light sources (2), the at least two adjacent light sources (2) preferably differing from each other in characteristics of light beams emitted by them, and configured to illuminate a biological sample (4) at different incident angles to reconstruct a single image of the biological sample using Fourier staggered imaging, wherein the characteristics of the light beam of each light source (2) are configured to match the sample (4) to increase contrast and / or color or material information content in the reconstructed single image of the sample (4), wherein one or more of the light sources comprises ultraviolet light.

[0084] In some embodiments, the present disclosure relates to an imaging system (100) for Fourier staggered imaging, comprising an array of light sources (2) according to the present disclosure, such as an array of UV light sources.

[0085] In some embodiments, the present disclosure relates to a method for performing Fourier staggered imaging, the method comprising: irradiating (S10) a biological sample at different incident angles by at least two adjacent light sources in a light source array, wherein the at least two adjacent light sources preferably differ from each other in characteristics of light beams emitted by them, and the characteristics of the light beam of each of the at least two light sources match the sample to increase the contrast in a single image of the sample to be reconstructed, wherein at least one adjacent light source is an ultraviolet light source; acquiring (S20) an image of the biological sample by a camera with the aid of the light beams from the at least two adjacent light sources, wherein the biological sample is characterized as being unstained; and reconstructing (S30) a single image of the sample by using the captured image.

[0086] It should be understood that all advantageous option changes in the modifications described herein and above with respect to the implementation of the light source array according to the first aspect can be equally applied to the implementation of the imaging system for Fourier stack imaging according to the second aspect and the implementation of the method according to the third aspect, and vice versa.

[0087] Example 1

[0088] Currently, biological samples such as blood smears or tissue sections analyzed under a microscope require staining to visualize important information such as cell nuclei. This problem represents a major obstacle on the road to further automation, increases costs, and can lead to significant inter-laboratory variability in diagnostic results.

[0089] In embodiments, the deficiencies in FMP are overcome by a microscopy method based on UV Fourier staging microscopy (FPM) that is capable of directly imaging biological samples such as blood smears and digital pathology samples without the need for staining. Embodiments include a method for digitally staining these images to provide clinicians with images similar to those taken of stained samples.

[0090] FPM is a novel computational microscopy technique that combines the ability to record images at high resolution with a large field of view. This is achieved by illuminating the sample from multiple directions at angles lower and higher than the numerical aperture of the objective used. The resulting low-resolution images are then stitched together in Fourier space using an iterative algorithm based on the Gerchberg-Saxton algorithm. Traditionally, FPM has been used with great success in the visible range of the spectrum. However, biological samples have little absorption in this spectral range. By shifting the wavelength from visible light to the UV, where the natural absorption bands for nucleotides and proteins are at approximately 260nm and 280nm, respectively, the absorption contrast of biological samples is greatly increased.

[0091] Example 2: UV-FPM for imaging biological samples without natural absorption contrast

[0092] Fourier stacking microscopy (FPM) is a computational microscopy technique that generates high-resolution images over a large field of view (FoV). This is achieved by acquiring images of the sample at various illumination angles larger than the numerical aperture (NA) of the objective used, and iteratively combining these images into a high-resolution image in Fourier space. The illumination angle is chosen so that the images overlap in Fourier space by about 40% (e.g. 35% to 45%) and depends on several parameters, such as the objective or detector used. The resolution limit of FPM microscopy is determined by the microscope objective used and the maximum illumination angle (α max ) and the refractive index of the medium (n):

[0093]

[0094] .

[0095] Equation 1

[0096] Currently, illumination in Fourier stacking microscopy is achieved by using arrays of visible light LEDs. Many designs of such arrays have been proposed, such as planar arrays, hemispherical designs. But in general, they all follow the same design concept: LEDs are attached to some kind of carrier plate, which is then placed at a certain distance from the sample. The LEDs are then flashed continuously to achieve illumination at a specific illumination angle. In an embodiment, such LEDs are placed with one or more light sources. The potential applications of this technology are wide-ranging. With a focus on diagnostics, FPM can replace standard optical microscopy. For example, hematology (especially blood smear analysis) or digital pathology. Such potential applications have gained significant interest in both academia and industry. For most diagnostic applications, the workflow can be summarized as follows. Take the sample, fix it on a carrier, such as a microscope slide, stain it, and then image it on the FPM microscope. Ideally, such a workflow is automated to increase throughput, reduce costs, and achieve consistent results. Staining is performed to produce the absorption contrast required to extract information from the microscope image. At the same time, staining represents a major obstacle because it leads to increased costs, it is also time-consuming, and the protocol can vary greatly between laboratories. The present disclosure provides a FPM-based method using unstained images or imaging. Due to the characteristics of image reconstruction based on the Geisberg-Saxon iterative phase retrieval algorithm, FPM reconstruction not only brings high-resolution amplitude images, but also brings the calculation of qualitative phase differences. There have been some suggestions to use this phase information to visualize objects that do not have natural absorption contrast. However, the phase differences displayed in FPM images today are not enough to distinguish white blood cells (WBC).

[0097] In embodiments, the present disclosure includes a UV-based FPM system that can image biological samples without the need for staining. Currently, samples are stained to produce the absorption contrast required to extract information from biological samples, such as identifying white blood cell nuclei. FPM-based systems are based on visible light, a frequency at which most biological samples show minimal absorption. As discussed in the previous section, this is problematic because the sample needs to be stained to produce the absorption contrast required for FPM imaging. Therefore, the present disclosure proposes shifting the wavelength range used for FPM. It is well known that nucleic acids and proteins absorb at 260nm and 280nm, respectively (see Figure 6 ). For example, 260 nm radiation is often used to quantify DNA. C=C double bonds also absorb in the UV, although the position of the absorption band depends on the amount of electron delocalization and can shift significantly. Figure 6 , Figure 6UV absorption of proteins and nucleic acids is described (from: World Wide Web: handling-solutions.eppendorf.com / sample-handling / photometry / applications / detailview-applications / news / uv-vis-spectrophotometry-easy-and-quick-quantification-of-nucleic-acids / ).

[0098] In addition to the natural absorption contrast, reducing the wavelength has the added benefit of increasing resolution at a constant numerical aperture (see Equation 1). This also means that a lower NA is required. sys to produce the same resolution as visible light FPM. This, in turn, means that either fewer LEDs are needed (at lower illumination angles) or a lower NA objective can be used to achieve the target resolution.

[0099] The following implementation focuses on the identification and classification of nuclei within blood smears and white blood cells, but the concept can be easily extended to other use cases where the sample has no absorption contrast in the visible light wavelength range. Another example is a tissue section that is stained to visualize the nuclei and other features within the sample.

[0100] Shifting the wavelength(s) used for FPM imaging to this wavelength range allows imaging of biological specimens without the need for staining. Fig. 7A and 7b Images of the same leukocyte measured once with visible light (approximately 450 nm) and once with UV light at 280 nm are shown. These images clearly show how the nucleus is only visible when imaged with UV light. Note that these images are single images without FPM reconstruction and therefore have a rather low resolution. Reference is now made to Fig. 7A and 7b , Magnified images of an unstained blood smear on a quartz slide imaged once at 7 (A) 600nm and 7 (B) 280nm. Images were taken with a single UVLED under the sample and a 10x / 0.2NA objective. A WBC is in the center of the image. The increased absorption contrast of the cell nucleus is evident when imaging at 280nm.

[0101] Reference now Figure 8 , Figure 8 Shown is a FPM reconstruction of a leukocyte when imaged at 280 nm. A total of 121 images were acquired at various illumination angles up to a maximum illumination angle of approximately 35° and stitched together in Fourier space to generate a high resolution image. Figure 8 FPM reconstruction of a blood smear image imaged at 280 nm is described.

[0102] It has been shown that biological specimens for FPM benefit from being embedded in a refractive index matching medium such as DPX mounting medium or microscope immersion oil and then sandwiched between two slides. In the case of UVFPM, these slides as well as the mounting medium need to be UV transmissive, preferably in the range of 250nm-320nm. An example of such a medium is silicone oil. One option to reduce the cost of using quartz as a sample carrier is to use an inverted FPM microscope. In this way, only a thin coverslip made of quartz is needed to minimize absorption. In this case, the sample can also be mounted directly on the coverslip without the need for a second slide.

[0103] Pathologists often analyze stained specimens. Therefore, they will preferably be presented with color images to drive acceptance. Digital staining of FPM images can be used to generate color FPM images. Such staining can be achieved by direct superposition of FPM images measured at multiple wavelengths and adjusting the white balance of the images or by training an AI algorithm to stain the images. For both staining methods, it is recommended to acquire UV-FPM images at at least 2 wavelengths. It is also recommended to use at least one wavelength in the non-absorption range of the sample as a contrast stain.

[0104] The ability to perform imaging of biological specimens at high resolution with a large FoV and without the need for staining also opens the door to increased automation of diagnostic workflows at reduced cost. It should be noted, however, that even with the large FoV provided by the FPM, scanning of the specimen may still be necessary. Images can also be stitched together to produce an image of the entire specimen, with the ability to zoom in and out simulating an optical microscope. In addition, the FPM allows for digital refocusing of the specimen. Paired with the ability to perform z-stacks, this will allow for tomographic imaging of the specimen.

[0105] As mentioned above, most commonly some form of LED array is used in FPM. The light source can be patterned so that different wavelengths are used at different locations and subsequently reconstructed into a single image. Polarization filters or polarization sensitive cameras can also be used for UV-FPM. This will minimize the effects of scattered background light and will bring about a reduction in noise. The concept of polarization FPM will be covered in more detail in a separate invention disclosure.

[0106] It should be noted that changing the wavelength to UV requires some changes to a standard FPM microscope. The LEDs (or any other light source used) will be replaced by their corresponding UV counterparts. Since UV LEDs are now readily available, this will not cause any greater problems. In addition, all optical components and cameras need to be UV compatible. This means, for example, changing all glass-based parts to quartz. The sample also needs to be mounted on a quartz microscope slide.

[0107] In general, the above concept can be extended to all situations where the sample is not observed in the visible range but in the UV range, such as digital pathology. Tissue sections are also stained to create absorption contrast within the sample and the FPM.

[0108] The main difference to known solutions is the use of UV light based FPM. Instead of using one or more stains to create absorption contrast not present in the sample, the wavelength of the light is changed to exploit naturally occurring absorption bands. This results in: easier automation, reduced hands-on time, elimination of variability due to different staining protocols, and easier comparison of results produced in different laboratories.

[0109] In the foregoing detailed description, various features are grouped together in one or more examples for the purpose of streamlining the present disclosure. It should be understood that the above description is intended to be illustrative rather than restrictive. It is intended to cover alternative modifications and equivalents. Many other examples will be apparent to those skilled in the art upon reviewing the above description.

[0110] Reference numerals list

[0111] 2 Light Source

[0112] 3 Carrier board

[0113] 4 samples

[0114] 5 Sample plane

[0115] 6 Objective lens

[0116] 7 Areas of Interest

[0117] 8 hollow rectangle

[0118] 10 Tube lens

[0119] 12 Camera

[0120] 14 Irradiation equipment

[0121] 16 base units

[0122] 18 hemispherical carrier

[0123] 20 circular sub-areas

[0124] 100 Fourier stack imaging system

[0125] S10-S30 Method Steps

Claims

1. An array of light sources (2) of an imaging system (100) for Fourier ptychography, the array of light sources comprising at least two adjacent light sources (2), the at least two adjacent light sources preferably differing from each other in the properties of the light beams emitted by them, and configured to illuminate a sample (4) at different incident angles to reconstruct a single image of the sample using Fourier ptychography, wherein: The characteristics of the light beam of each light source (2) are configured to match the sample (4) to increase contrast and / or color or material information content in the reconstructed single image of the sample (4).

2. The light source array according to claim 1, wherein: The characteristics of the light beam include the wavelength, bandwidth, spectral curve and / or polarization of the light beam.

3. The light source array according to claim 1 or 2, wherein: The contrast in the reconstructed single image of the sample (4) depends on the interaction characteristics of the light beam of the corresponding light source (2) with a given position of the sample (4).

4. The light source array according to claim 3, wherein: The interaction characteristics include absorption, refraction, scattering, diffraction and / or fluorescence of the light beam of the corresponding light source (2) at the given position of the sample (4).

5. The light source array according to claim 3 or 4, wherein: The interaction characteristics depend on the characteristics of the light beams of the respective light sources (2) and the material and / or geometry of the sample (4) at the given location.

6. A light source array according to any one of the preceding claims, wherein: The light source (2) array comprises at least three light sources (2), the three light sources being a first light source, a second light source and a third light source, wherein the first light source is adjacent to the second light source, the second light source is further adjacent to the third light source, wherein the first light source and the second light source are different from each other in characteristics of light beams emitted by them, wherein the second light source and the third light source are different from each other in characteristics of light beams emitted by them, and wherein the first light source and the third light source include the same characteristics from the light beams emitted by them.

7. The light source array according to claim 6, wherein: In the reconstruction process using Fourier phantom imaging, the images captured by means of the second light source include lower weighting factors than the images captured by means of the first light source and the third light source.

8. The light source array according to claim 6, wherein: In the reconstruction process using Fourier phantomography, the image captured by means of the second light source includes the same weighting factors as the images captured by means of the first and third light sources.

9. A light source array according to any one of the preceding claims, wherein: The light source (2) array comprises a plurality of light sources (2), in particular more than three light sources (2), preferably more than five light sources (2), wherein the plurality of light sources form at least two clusters, wherein the light sources within each cluster are adjacent to each other, and wherein the characteristics of the light beams of the light sources within each cluster are configured to match a given position of the sample (4) so ​​as to increase the contrast in the reconstructed single image of the given position of the sample (4).

10. A light source array according to any one of the preceding claims, wherein: The light source array is configured to be selected based on the type of the sample (4).

11. An imaging system (100) for Fourier stack imaging, comprising an array of light sources (2) according to any one of claims 1 to 10.

12. The imaging system (100) of Fourier stack imaging according to claim 11, comprising a bandpass filter, a notch filter, a filter defining a composite spectral curve and / or an edge filter arranged between a camera sensor (12) of the imaging system (100) of Fourier stack imaging and the light source (2) array.

13. The imaging system (100) of Fourier stack imaging according to claim 11 or 12, wherein: The imaging system (100) of Fourier stack imaging comprises an objective lens (6), wherein the objective lens aperture of the objective lens (6) can be divided into a plurality of segments, wherein each segment of the objective lens aperture is associated with a light source (2) or a cluster of light sources (2) in the light source (2) array.

14. A method of performing Fourier phantom imaging, comprising: illuminating (S10) the sample at different angles of incidence by at least two adjacent light sources in an array of light sources, wherein the at least two adjacent light sources preferably differ from each other in properties of the light beams emitted by them, and the properties of the light beam of each of the at least two light sources are matched to the sample to increase contrast in a single image of the sample to be reconstructed; acquiring ( S20 ) an image of the sample by a camera using the light beams from the at least two adjacent light sources; and The single image of the sample is reconstructed ( S30 ) by using the captured image.

15. The method according to claim 14, wherein: The characteristics of the light beam include the wavelength, bandwidth and / or polarization of the light beam.

Images