Specimen mount for microscope

Abstract

The sample mount (10) for imaging at least one sample (50) includes a hydrogel body (25) and at least one cavity (30). The cavity (30) is disposed in the hydrogel body (25) for holding the at least one sample (50) in a culture environment (60). The at least one cavity (30) has an opening (25b) that provides access to the at least one cavity (30) for placing the at least one sample (50) in the at least one cavity (25) and for providing culture medium and / or gas to the at least one sample (50).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to European Patent Application No. 23162464.4, the disclosure of which is hereby incorporated in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to sample mounts for microscopy, and in particular to sample mounts for culturing and imaging living samples. [Background technology]

[0003] Embryo implantation is a crucial developmental stage in mammalian species that relies on embryo-maternal interactions. For example, the placenta provides nutritional support for embryonic development. However, the inaccessibility of the implanted embryo within the uterus by experimental techniques, such as light-sheet microscopy, makes it difficult to investigate the role of embryo-uterine interactions in peri-implantation development.

[0004] Another approach is based on investigating embryo-uterus interactions in vitro by placing samples, e.g., embryos, in a synthetic environment. One example of a synthetic environment is an in vitro environment that provides samples with (bio)engineered conditions that resemble the biochemical and mechanical conditions in vivo. However, there is a lack of (bio)engineered uterus-like microenvironments that provide conditions sufficiently similar to the natural environment of embryos in the uterus.

[0005] There is a need for a synthetic environment for imaging living samples (e.g., organoids, tissue explants, or embryos) to provide quantitative data with high temporal and spatial resolution and simultaneously reduce the burden of illumination light on the sample.

[0006] An example of a synthetic environment is proposed by Bondarenko et al. (bioRxiv, 2022, DOI:10.1101 / 2022.06.13.495767). They describe a uterus-like microenvironment designed to replicate mouse embryonic development in vitro up to E5.25. This artificial microenvironment allows imaging of developing mouse embryos using light-sheet microscopy.

[0007] US Pat. No. 11,155,775 B2 discloses a sample holder made of hydrogel that surrounds a culture gel in which cells or tissues are embedded. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure relates to a sample mount for imaging a living sample. In particular, the present disclosure relates to a sample mount for imaging a living sample, comprising a three-dimensional structured hydrogel. The present disclosure further relates to a method for manufacturing the sample mount. The present disclosure further relates to a method for imaging a living sample. At least one sample may be a mammalian sample. The at least one sample may comprise a single cell or a group of cells. The group of cells may be a cell cluster, for example, a developing embryo, an organ, or a tissue. The cell cluster may be an embryo model comprising pluripotent stem cells, embryonic cells, and / or extraembryonic cells. The group of cells may be, for example, an organoid prepared in vitro, or a tissue sample obtained, for example, from a biopsy.

[0009] The sample mount for imaging at least one sample includes a hydrogel body and at least one cavity. The at least one cavity has an opening that provides access to the at least one cavity. The opening is suitable for placing the at least one sample in the at least one cavity and for providing a culture medium and / or gas to the at least one sample. The at least one cavity is disposed in the hydrogel body to hold and / or position the at least one sample in a culture environment. The at least one cavity provides structural support for the at least one sample.

[0010] By providing a sample holder with a hydrogel body, it is possible to provide a biocompatible sample holder with biochemical properties that promote sample culture and / or sample development, e.g., growth. Thus, the hydrogel body contributes to the culture environment in which the sample is placed. Furthermore, a biocompatible sample holder including a hydrogel body allows for the supply of nutrients to at least one sample on the hydrogel body. Furthermore, the hydrogel body allows for the formation or molding of at least one cavity in the hydrogel body, the cavity having geometric properties that mimic the geometrical conditions and mechanical properties of the at least one sample's native environment.

[0011] The culture environment may include at least one gas (e.g., oxygen, nitrogen, and / or carbon dioxide) and / or a liquid culture medium. Providing the at least one gas allows for providing atmospheric conditions for the at least one sample in the culture environment that allow for the exchange of the at least one gas with the at least one sample and / or liquid culture medium. The liquid culture medium allows for the cultivation, i.e., growth and / or nutrient supply, of the at least one sample.

[0012] The at least one cavity may be dimensioned to provide mechanical support, for example, by adhering and / or contacting the at least one sample to the wall of the cavity formed in the hydrogel body. Adhering and / or contacting the at least one sample to the wall of the at least one cavity allows for mimicking the at least one sample's natural environment. The adhesion and / or contact provides structural support to the at least one sample. The adhesion and / or contact also reduces drift of the at least one sample in the at least one cavity, thereby enabling positioning of the at least one sample. This positioning allows for tracking of individual samples over the course of one or more measurements.

[0013] The at least one cavity may be dimensioned to provide an extracellular matrix (ECM) for the at least one sample, the extracellular matrix enabling attachment of the at least one sample to the at least one cavity, biochemical communication between cells within the at least one sample, biochemical communication between cells and the ECM, differentiation of the at least one sample, development of the at least one sample, and / or growth of the at least one sample.

[0014] In one embodiment, the sample mount may include multiple cavities, the multiple cavities being arranged along the first direction. A sample mount according to a further embodiment allows for multiple samples to be provided on one sample mount.

[0015] In another embodiment, the plurality of cavities may be arranged three-dimensionally, for example, the plurality of cavities may be arranged in a three-dimensional array.

[0016] The hydrogel body may comprise one or more hydrogels, hi one embodiment, the one or more hydrogels may be arranged in one or more layers.

[0017] The hydrogel body has one or more components. The one or more components may be derived from animal, plant, and / or fungal sources, and / or chemically synthesized sources. The one or more components may include one or more of amino acids, polypeptides, proteins, nucleotides, oligonucleotides, nucleic acids, carbohydrates, lipids, vitamins, choline, and / or minerals. The one or more components may include one or more nutrients. The nutrients may be selected depending on the at least one sample to be imaged. The one or more components may include biological cells. The hydrogel body may be made from animal, plant, and / or fungal sources, and / or chemically synthesized sources. The one or more components enable the fabrication of a sample holder that enables the culturing and imaging of the at least one sample. The one or more components enable the provision of nutrients to the at least one sample on the one or more components of the hydrogel body. The one or more components enable the provision of an extracellular matrix to the at least one sample.

[0018] The hydrogel body may include, as one or more components, one or more of laminin, nidogen, collagen, glycoproteins, and / or proteoglycans.

[0019] The hydrogel body may include agarose as one or more components.

[0020] The hydrogel body, made from chemically synthesized sources, contains polyethylene glycol as one or more components.

[0021] The hydrogel body may comprise multiple polymers. In one aspect, the hydrogel body may be made from a blend of multiple hydrogels comprising multiple hydrogels. The multiple hydrogels may have different properties among the multiple hydrogels. The multiple polymers may, for example, have different chemical structures. The hydrogel blend may comprise a fibrous matrix.

[0022] In another embodiment, multiple hydrogels may be arranged in one or more layers, which can provide a spatial gradient of physicochemical properties.

[0023] The hydrogel body may be crosslinked and / or coated with a peptide (e.g., arginylglycylaspartic acid) and / or one or more components found in naturally occurring extracellular matrix (e.g., laminin, collagen, proteoglycans, glycoproteins, and / or enzymes), which crosslinks and / or coats the hydrogel body to provide at least one sample with an extracellular matrix.

[0024] The physicochemical properties of the hydrogel body can be manipulated by at least one of enzymatic degradation, non-enzymatic reaction, UV light irradiation treatment, and / or infrared light (IR) irradiation treatment, thereby providing an extracellular matrix to at least one sample.

[0025] The at least one cavity may extend along the second direction or be inclined relative to the second direction. When the second direction is vertical, this orientation of the at least one cavity allows easy access to the at least one cavity from above to place the at least one sample in the cavity, and / or allows access and / or manipulation of the culture environment from above. Furthermore, this orientation of the at least one cavity allows, for example, the use of gravity to retain a liquid medium in the at least one cavity.

[0026] The sample mount may further include a connector attachable to the sample mount, and the sample mount (10) attached to the connector may be arranged in a sample mount stack containing multiple sample mounts. Stacking multiple sample mounts in a sample mount stack allows for increased efficiency and throughput in analyzing the samples in the sample mounts.

[0027] The connector may be configured to fluidly connect the sample mount with a culture medium and / or gas to provide a culture environment, whereby the culture medium and / or gas can be provided to the sample mounts arranged in the sample mount stack.

[0028] A microscope for imaging a sample includes at least one illumination lens for illuminating the at least one sample, at least one detection objective for detecting light emitted or reflected by the at least one sample, and a sample mount according to the present disclosure. The at least one illumination lens may be an illumination objective. The at least one illumination objective has an illumination optical axis. The at least one detection objective has a detection optical axis. The detection optical axis may be disposed perpendicular to the illumination optical axis. In another embodiment, the detection optical axis may be disposed non-perpendicular to the illumination optical axis.

[0029] In one embodiment, one of the at least one illumination objective and one of the at least one detection objective may form a single objective. In this embodiment, the single objective serves both to illuminate the at least one sample and to detect light emitted or reflected by the at least one sample. In this embodiment, the illumination optical axis and the detection optical axis of the single objective may be arranged non-perpendicular to each other. For example, the angle between the illumination optical axis and the detection optical axis of the single objective may be less than 90 degrees. For example, the angle may be an acute angle.

[0030] The microscope may include additional elements, such as a camera, filters, additional lenses, and / or mirrors (not shown). For the sake of brevity, these additional elements will not be described in detail here. The camera may be a CCD detector or a CMOS detector. In embodiments of the present disclosure, the microscope may further include a control system that controls illumination and incubation based on at least one image acquired from at least one sample.

[0031] The microscope may further include elements for one or more of refocusing, translating, tilting, and / or rotating the illumination optical axis. Additionally, the microscope may further include elements for one or more of refocusing, translating, tilting, and / or rotating the detection optical axis. In one embodiment, the detection optical axis may be positioned to coincide with the illumination optical axis. In another embodiment, the detection optical axis may be positioned perpendicular to the illumination optical axis (as described above).

[0032] The microscope may be a light sheet microscope in which at least one sample is illuminated by a light sheet. The light sheet, oriented along an illumination optical axis, may be arranged perpendicular or non-perpendicular to a detection optical axis. Multiple illumination optical axes may be used to simultaneously or sequentially illuminate at least one sample from multiple illumination directions. Multiple detection optical axes may be used to simultaneously or sequentially detect light emitted or reflected from at least one sample in multiple detection directions.

[0033] The microscope may enable three-dimensional (3D) imaging of at least one sample by one or more of: moving a sample mount in at least one sampling direction; rotating the sample mount; moving one or more of at least one illumination objective, at least one detection objective, and a single objective; and / or moving and / or rotating a light sheet.

[0034] The microscope may also include other types of light sheet microscopes, such as microscopes using oblique plane microscopy (OPM). Oblique plane microscopy is a light sheet microscopy technique that uses a single high-numerical aperture microscope objective to both illuminate an oblique plane within at least one sample and collect fluorescence from the obliquely illuminated plane. Including corrective optics between the main microscope objective and the camera enables imaging of the oblique plane within at least one sample.

[0035] The microscope may have a different configuration to the one described above, e.g., a tilted illumination optical axis, an objective with a tilted optical axis, or a tilted light sheet, and light sheet microscopy has been implemented with a perpendicular illumination / detection scheme.

[0036] The plurality of cavities may be movable along at least one sampling direction, allowing for increased efficiency and throughput in analyzing samples in the sample mount.

[0037] In one embodiment, the multiple cavities are movable in three sampling directions, which allows for imaging of multiple samples placed in the multiple cavities, for example, when the multiple cavities are arranged in three dimensions, such as in a three-dimensional array.

[0038] A method for fabricating a specimen mount for a microscope includes providing a moldable hydrogel in a mold, placing a stamp within the moldable hydrogel, curing the hydrogel to form a hydrogel body with the mold and at least one cavity with the stamp, and removing the stamp from the cured hydrogel body.

[0039] The method may further comprise treating a surface of the at least one cavity.

[0040] The hydrogel may be a liquid hydrogel.

[0041] In another embodiment, moldable hydrogels may be produced from milled hydrogel particles. The milled hydrogel particles may be mixed with water with or without a catalyst (e.g., enzyme molecules, ions). Adding a catalyst allows for adjusting the pH, ion concentration, or enzyme activity to promote polymerization of the hydrogel particles. Mixing the milled hydrogel particles with water produces a moldable hydrogel. Mixing the milled hydrogel particles with water results in gelation, yielding a moldable hydrogel.

[0042] A method for imaging at least one sample with a microscope includes providing at least one sample in at least one cavity of a sample mount, providing an incubation environment in the at least one cavity, illuminating the at least one sample with at least one illumination objective having an illumination optical axis, and detecting light emitted or reflected by the at least one sample with at least one detection objective having a detection optical axis, wherein the detection optical axis is positioned perpendicular or non-perpendicular to the illumination optical axis. The method further includes acquiring at least one image of the sample.

[0043] The method for imaging at least one sample may further include moving the sample mount in at least one sampling direction, the at least one sampling direction being perpendicular to the detection optical axis and the illumination optical axis. The method for imaging at least one sample allows for imaging of the sample in a culture environment that allows for growth and development of the at least one sample. Furthermore, this method allows for maintaining and / or adjusting the culture environment while imaging the at least one sample. Furthermore, this method allows for high-throughput imaging. [Brief explanation of the drawings]

[0044] [Figure 1a] FIG. 1a illustrates a cross-sectional view of a sample mount for imaging a sample, according to one embodiment of the present disclosure. [Figure 1b] FIG. 1b shows a cross-sectional view of a sample mount for imaging a sample in a microscope according to another embodiment. [Figure 2] FIG. 2 shows an enlarged cross-sectional view of the cavity. [Figure 3a] FIG. 3a illustrates a top view of a sample mount according to one embodiment. [Figure 3b] FIG. 3b shows a top view of a sample mount according to a further embodiment. [Figure 4a] FIG. 4a illustrates a side view of a sample mount positioned on a microscope for imaging a sample, according to one embodiment. [Figure 4b] FIG. 4b shows a side view of a sample mount positioned on a microscope for imaging a sample, according to another embodiment. [Figure 4c] FIG. 4c shows a side view of a sample mount positioned relative to an objective unit of a microscope for imaging a sample, according to a further embodiment. [Figure 4d] FIG. 4d shows a side view of a sample mount positioned relative to a single objective lens of a microscope for imaging a sample, according to yet a further embodiment. [Figure 4e] FIG. 4e illustrates a side view of a sample mount positioned relative to a single objective lens of a microscope for imaging a sample, according to another embodiment. [Figure 5a] FIG. 5a illustrates a top view of a sample mount positioned on a microscope for imaging a sample, according to another embodiment. [Figure 5b] 5b shows a cross-sectional view of two of the sample mounts of FIG. 1 arranged in a sample mount stack. [Figure 6] FIG. 6 shows a perspective view of a sample mount according to a further embodiment. [Figure 7] FIG. 7 shows a perspective view of a sample mount according to a further embodiment. [Figure 8] FIG. 8 is a diagram showing a schematic diagram of a medium exchange unit. [Figure 9] FIG. 9 shows a flow chart of a method for manufacturing a sample mount. [Figure 10] FIG. 10 shows a mold for forming at least one cavity in the hydrogel. [Figure 11] FIG. 11 shows a flow chart of a method for imaging a sample. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will now be described with reference to the drawings. It will be understood that the embodiments and aspects of the present invention described herein are merely examples and do not limit the scope of protection of the claims in any way. The present invention is defined by the claims and their equivalents. It will be understood that the features of one aspect or embodiment of the present invention can be combined with the features of one or more different aspects and / or embodiments of the present invention.

[0046] FIG. 1a shows a cross-sectional view of a specimen mount 10. The specimen mount 10 facilitates imaging of at least one specimen 50 in a microscope 80. The specimen mount 10 particularly facilitates long-term live imaging of cell clusters and / or macroscopic tissue samples. Such tissue samples may be, for example, pre-implantation or post-implantation mammalian embryos, organoids, and / or tissue explants. The cell clusters may be embryonic models comprising pluripotent stem cells, embryonic cells, and / or extraembryonic cells.

[0047] The specimen mount 10 includes a hydrogel body 25. The hydrogel body 25 may be transparent, particularly to light used in fluorescence microscopy, such as light sheet microscopy. The hydrogel body 25 may have a stiffness in the range of approximately 1 Pa to 10 MPa.

[0048] In the embodiment shown in FIG. 1a, surface 25a of hydrogel body 25 is horizontal. Surface 25 may also be substantially planar. However, surface 25a of hydrogel body 25 is not limited to being horizontal or substantially planar. Surface 25a may have a non-horizontal surface or a non-planar surface. In one embodiment, surface 25a may face vertically upward (see, e.g., FIGS. 4a-4c). In another embodiment, surface 25a may face in a different direction, such as horizontally or downward. In still further embodiments, hydrogel body 25 may have multiple surfaces 25a. Some of the multiple surfaces 25a may face in different directions from each other.

[0049] The sample mount 10 further includes at least one cavity 30. The at least one cavity 30 is disposed in the hydrogel body 25. The at least one cavity 30 is arranged to hold a sample 50. The at least one cavity 30 includes walls 70 for providing a contact or adhesive surface for the at least one sample 50. The contact or adhesive surface includes the walls 70. The contact or adhesive surface may further include a bottom / end in the direction of the cavity axis (see below) of the at least one cavity 30. The contact or adhesive surface may have a non-planar shape.

[0050] At least one cavity 30 has an opening 25b. The opening 25b provides access to the at least one cavity 30, thereby enabling access to at least one sample 50 placed in the at least one cavity 30, for example, for fluid communication with the at least one sample 50 or for placing the at least one sample 50 in the at least one cavity 30. The opening 25b may be provided on the surface 25a. The at least one sample 50 can be placed in the cavity 25 through the opening 25b. The at least one sample 50 can be provided with a culture medium and / or a gas. The gas can be provided through the opening 25b or by diffusion into the at least one cavity 30. The at least one cavity 30 may contain a culture medium and / or a gas.

[0051] The at least one cavity 30 may be configured to provide a culture environment 60 for the at least one sample 50. The culture environment 60 may include a culture medium and / or a gas. The culture environment 60 may provide an extracellular matrix (not shown) to provide mechanical and / or biochemical support for the at least one sample 50. In one embodiment, the extracellular matrix is ​​a synthetic extracellular matrix. The extracellular matrix (ECM) provides an environment that allows one or more of: attachment of the at least one sample 50 to the wall 70; intercellular biochemical communication within the at least one sample 50; biochemical communication between cells of the at least one sample 50 and the ECM; differentiation of the at least one sample 50; development of the at least one sample 50; and / or growth of the at least one sample 50.

[0052] The hydrogel body 25 extends along a first direction A1, a second direction A2, and a third direction A3. Thus, the hydrogel body 25 has a length extending along the first direction A1, a height extending along the second direction A2, and a width extending along the third direction A3. The first direction A1, the second direction A2, and the third direction A3 form a three-dimensional Cartesian coordinate system. The first direction A1 and the third direction A3 are, for example, horizontal directions. The second direction A2 is, for example, vertical directions. In one embodiment, the Cartesian coordinate system may be aligned with, for example, the surface 25a of the hydrogel 25, which is positioned horizontally, and the cavity axis CA of at least one cavity 30, which is positioned vertically (see FIGS. 1b and 2). In another embodiment, the Cartesian coordinate system may be defined by an illumination optical axis IA and / or a detection optical axis DA (see below).

[0053] The hydrogel body 25 includes one or more components. The hydrogel body 25 may include at least one polymer. The polymer may be a synthetic polymer, such as, but not limited to, polyvinyl alcohol, polyethylene glycol (PEG), sodium polyacrylate, acrylate polymers, and copolymers thereof. The polymer may be a natural polymer, such as, but not limited to, hyaluronic acid, chitosan, heparin, alginate, and fibrin. The hydrogel body 25 may be made from an animal source. The animal source may include one or more of laminin, nidogen, collagen, glycoprotein, proteoglycan, and / or enzyme. The hydrogel body 25 may be made from a plant source and / or a fungal source. The plant source and / or a fungal source may include agarose.

[0054] In one embodiment, at least one polymer of the hydrogel body 25 may be crosslinked or chemically coupled via non-covalent bonds. The polymer of the hydrogel body 25 may be crosslinked with one or more peptides. The one or more peptides may include, for example, metalloprotease-cleavable peptides to facilitate interaction between the sample 50 and the walls of the at least one cavity 30. In particular, the hydrogel 25 of the at least one cavity 30 may be crosslinked in this manner.

[0055] In another embodiment, the hydrogel body 25 may be functionalized with one or more peptides, one example of which is RGDSPG (Arg-Gly-Asp-Ser-Pro-Gly).

[0056] In further embodiments, at least one polymer of the hydrogel body 25 may be coated with one or more of peptides (e.g., arginylglycylaspartic acid) and / or extracellular matrix components (e.g., laminin, collagen, proteoglycans, and / or glycoproteins), etc. In particular, the wall 70 of at least one cavity 30 may be so coated.

[0057] In yet further aspects, the hydrogel body may comprise multiple polymers. The hydrogel body may be made from a hydrogel blend comprising multiple hydrogels. The multiple hydrogels may have different properties among the multiple hydrogels. The multiple polymers may, for example, differ in chemical structure.

[0058] The hydrogel body 25 may include a fibrous matrix. The fibrous matrix may be formed based on one or more components of the hydrogel body 25. The fibrous matrix may be formed from a polymer derived from, for example, a combination of collagen with PEG and optionally Matrigel, a combination of Matrigel with collagen, or one or more of PEG, fibrin, and a polymer suitable for 3D printing of a fibrous matrix that is compatible with at least one sample 50. The fibrous matrix may be formed from multiple polymers. The fibrous matrix provides structural support for at least one sample 50, such as a cell cluster or tissue sample, held by the sample holder during growth. Examples of cell clusters or tissue samples are mouse embryos or human embryos. The cell cluster may be an embryo model including pluripotent stem cells, embryonic cells, and / or extraembryonic cells.

[0059] During fabrication, multiple hydrogels can be deposited in one or more layers (not shown). The physicochemical properties of hydrogel body 25 can vary among some of the one or more layers. Examples of physicochemical properties include one or more concentrations of one or more components of hydrogel body 25, one or more stiffnesses of one or more layers, one or more shapes of one or more layers, etc.

[0060] In one embodiment of the present disclosure, one or more hydrogels may be optimized for the growth of at least one sample 50, e.g., one or more cells, cell clusters, organoids, tissues, or embryos. The cell clusters may be embryo models including pluripotent stem cells, embryonic cells, and / or extraembryonic cells. One or more components of the one or more hydrogels may include one or more types of biological cells. For example, in the context of in vitro mammalian embryogenesis, e.g., in vitro mouse embryogenesis, uterine cells such as endometrial cells and stromal fibroblasts may be added to the one or more hydrogels (e.g., with PEG, collagen, and / or Matrigel) to support embryo growth. In the context of organogenesis, stromal cells surrounding developing organs and / or developing organoids may be added to the one or more hydrogels. In another embodiment, the one or more hydrogels may include blood cells and / or immune cells, i.e., macrophages and / or leukocytes, for live imaging of multicellular interactions in reduced physiological systems. Other types of biological cells may be mast cells and / or red blood cells. The biological cells may be mixed with one or more hydrogels. The biological cells may be added to one or more hydrogels during the manufacture of the sample mount 10. Alternatively, the biological cells may be deposited on the surface 25a of the hydrogel body and / or on the wall 70 of at least one cavity 30.

[0061] The physicochemical properties of one or more hydrogels, such as one or more stiffnesses or one or more shapes, can be configured and / or dynamically adjusted by at least one of enzymatic degradation, non-enzymatic reactions, ultraviolet (UV) irradiation treatment, infrared (IR) irradiation treatment, and / or laser treatment. Enzymatic degradation includes adjusting the physicochemical properties of one or more hydrogels using peptidases, such as metalloproteases. The peptidases may be provided externally using additional medium (with or without perfusion of the hydrogel matrix) or internally by the sample itself. In another embodiment, the peptidases may be provided with the culture medium. Non-enzymatic degradation may include engineering one or more hydrogels to be capable of hydrolysis. UV irradiation treatment may include using UV irradiation to polymerize or depolymerize one or more hydrogels. IR irradiation treatment may include polymerizing or depolymerizing one or more hydrogels. Laser treatment may involve using laser light to locally manipulate the physicochemical properties of one or more hydrogels in hydrogel body 25, for example, using two-photon depolymerization, thereby enabling three-dimensional structuring of hydrogel body 25. Laser treatment may involve diffusing cross-linked molecules, such as growth factors and signaling molecules, from hydrogel body 25 into a treatment medium.

[0062] In one embodiment, the sample mount 10 may include a housing 20. The housing 20 may house the hydrogel body 25. The housing 20 may be disposed on the surface of the hydrogel body 25. In one embodiment, the housing 20 may be made of a different material than the hydrogel body 25. In another embodiment, the housing 20 may be made of a similar or identical material as the hydrogel body 25. In one embodiment of the present disclosure, the housing 20 may serve as a mold during the manufacturing of the sample holder 10.

[0063] In one embodiment, the housing 20 may be fabricated from a transparent material. One example of a transparent material used to manufacture the housing 20 is PDMS (polydimethylsiloxane). In one embodiment of the present disclosure, the transparent material used to manufacture the housing 20 is selected to have a refractive index that matches the refractive index of the hydrogel body 25 and / or the surrounding immersion medium (e.g., PBS, water). Furthermore, the transparent material may have a thickness of <100 μm. The transparent material may include, for example, fluorinated ethylene propylene (FEP) tubing / foil, polytetrafluoroethylene (PTFE), and / or tetrafluoroethylene (THV). The shape of the hydrogel body 25 may be configured so that the surface of the housing 20 is positioned perpendicular to the illumination optical axis IA of the illumination objective 85 (see, e.g., FIG. 4c). Furthermore, the shape of the hydrogel body 25 may be configured so that the housing 20 is positioned perpendicular to the detection optical axis DA of the detection objective 90 (see, e.g., FIG. 4c).

[0064] In one embodiment of the present disclosure, the housing 20 can be used to provide gases, such as oxygen, nitrogen, and / or carbon dioxide, and / or liquid culture medium. In this embodiment, the housing 20 may be sealable. Furthermore, in this embodiment, the housing 20 may include a supply space 65 for providing culture medium and / or gas to the at least one cavity 30 via the opening 25b. The supply space 65 may be part of the culture environment 60. The supply space 65 is disposed above the hydrogel body 25. The supply space 65 is fluidly connected to the at least one cavity 30 and allows the supply of culture medium and / or gas to the at least one sample 50 held in the at least one cavity 30. The culture medium and / or gas may be provided to the supply space 65 by a perfusion unit 40 (see FIG. 8 ) and / or directly to the hydrogel body 25. The perfusion unit 40 may include a microfluidic unit (not shown). The microfluidic unit may include one or more fluid-connecting microtubes PX (see FIG. 5b ). The microfluidic unit may fluidly connect one or more of the sample holder 10, the supply space 65, and the at least one cavity 30 to the perfusion unit 40. The microfluidic unit may further include a microchannel 31 (see below). The perfusion may be fluidly connected to the supply space 65 of the housing 20. The perfusion unit, fluidly connected to the housing 20, serves to exchange culture medium and / or gas. The perfusion unit may be fluidly connected to the housing 20, for example, via tubing (not shown). The exchange of culture medium and / or gas allows for long-term culturing of the at least one sample 50. The long-term culturing of the at least one sample 50 allows for long-term imaging of the at least one sample 50.

[0065] FIG. 1b shows a cross-sectional view of a sample mount 10 for imaging a sample 50 with a microscope 80 according to a further embodiment of the present disclosure. In this embodiment, the cavity 30 is inclined with respect to the second direction A2. The angle α between the negative second direction A2 and the cavity axis CA may be, for example, 90° (ninety degrees) or less. For example, the angle α may be 60° (sixty degrees) or less. In another embodiment, the angle α may be 45° (forty-five degrees) or less. However, the angle α may be equal to 90° (i.e., aligned with a horizontal cavity axis CA) or even greater than 90°. The angle α may depend on the design of the mold 28 (see below), the dimensions of the at least one cavity 30, and the design of the housing 20. The tilted arrangement of at least one cavity 30 shown in FIG. 1b is useful for inverted light sheet microscopy, in which the illumination optical axis IA of the illumination objective 85 is arranged at an illumination angle relative to the horizontal, and / or the detection optical axis DA of the detection objective 90 is arranged at a detection angle relative to the horizontal.

[0066] FIG. 2 shows an enlarged cross-sectional view of one embodiment of a cavity 30 according to one embodiment of the present disclosure. The cavity 30 extends along a cavity axis CA. The cavity axis CA may be parallel to the second direction A2. The cavity 30 includes at least a first section 30a and a second section 30b. The first section 30a and the second section 30b may be adjacently disposed along the cavity axis CA. In another embodiment, at least one cavity 30 may have more than two sections. In still further embodiments, at least one cavity 30 may have a shape different from that shown in FIG. 2. For example, at least one cavity 30 may have a well-like shape. In another embodiment, the shape of at least one cavity 30 may deviate from the linear cavity axis CA as shown in FIG. 2. For example, the shape and / or cavity axis CA of at least one cavity 30 may be curved.

[0067] The first compartment 30a is, for example, substantially cylindrical. The first compartment 30a has a diameter D1 and a length L1. In one embodiment of the present disclosure, the diameter D1 of the first compartment 30a is preferably 100 μm or more and preferably 160 μm or less. The length L1 of the first compartment 30a is preferably 150 μm or more and preferably 250 μm or less. These example dimensions allow for the culture and imaging of, for example, mouse embryos. If the at least one sample 50 is of a different type, the dimensions may differ from those shown above. For example, in an embryo in a post-implantation developmental stage, the dimensions of the at least one cavity may need to be up to 1 millimeter or more. In this case, the at least one cavity 30 may be formed across most of the hydrogel body 25, and the hydrogel body 25 may have a thinner structure to provide contact and / or adhesion.

[0068] The second compartment 30b is, for example, substantially conical. The second compartment 30b includes a first diameter D21, a second diameter D22, and a length L2. In one embodiment of the present disclosure, the first diameter D21 of the second compartment 30b is preferably 100 μm or more and preferably 160 μm or less. The second diameter D22 of the second compartment 30b is preferably 30 μm or more and preferably 90 μm or less. The length L2 of the second compartment 30b is preferably 300 μm or more and preferably 500 μm or less. The dimensions exemplified herein allow for the culture and imaging of, for example, organoids or implantation-stage mammalian embryos, such as human or mouse embryos. Examples of organoids are intestinal crypts, glandular crypts, or endometrium.

[0069] The cavity 30 may further include a third compartment 30c. The third compartment 30c has a second diameter D22 and a length L3. The length L3 of the third compartment 30c is preferably 10 μm or more and preferably 50 μm or less. These example dimensions allow for the culture and imaging of, for example, one or more cyst-forming organoids, implantation-stage mouse embryos, or stem cell embryo models.

[0070] The at least one cavity 30 is dimensioned to allow adhesion of the sample 50 to the wall 70 of the at least one cavity 30. Adhesion of the at least one cavity 30 to the wall 70 may be mediated by cell adhesion molecules, such as cadherins, selectins, or integrins, which belong to the class of transmembrane proteins and are located in the cell membrane of the at least one sample 50. Cell adhesion molecule-mediated adhesion is a biochemical adhesion in which cell adhesion molecules bind to ligands such as glycoproteins (e.g., fibronectin or vitronectin), collagen, and laminin. The adhesion may be three-dimensional. The term "three-dimensional adhesion" should be understood to mean that when the at least one sample 50 is held in the at least one cavity 30, the contact points between the at least one sample 50 and the contact surface (e.g., wall 70) of the at least one cavity 70 are located on non-planar portions of the contact surface. The non-planar portions of the contact surface may be formed, for example, by local degradation and / or invasion of the hydrogel body 25 by the sample 50. The term "three-dimensional adhesion" should also be understood to mean mechanical contact between the at least one sample 50 and the wall 70 of the at least one cavity 30, thereby allowing the growth and / or cultivation of the at least one sample. The at least one cavity 30 is dimensioned to provide an extracellular matrix for the sample. The at least one cavity 30 is configured and dimensioned to mimic the biochemical and mechanical properties of natural uterine tissue, for example, for in vitro embryo development. The at least one cavity 30 may also be configured and dimensioned to mimic the biochemical and mechanical properties of the natural environment of other cell types, cell cultures, and / or organoids.

[0071] 3a shows a top view of the sample mount 10 according to one embodiment of the present disclosure. The surface 25a of the hydrogel body 25 may have, but is not limited to, a rectangular shape.

[0072] In the embodiment of sample mount 10 shown in FIG. 3 a, a plurality of cavities 30 are disposed in hydrogel body 25 .

[0073] The sample mount 10 shown in FIG. 3a includes three cavities 30. However, the number of cavities 30 may vary. For example, the sample mount 10 may include 50 or 500 cavities 30. The cavities 30 are arranged along a first direction A1. In one embodiment of the present disclosure, the cavities 30 are further arranged along a third direction A3. In this embodiment, the dimensions of the cavities 30 and the dimensions of the optical arrangement of the at least one illumination objective 85 and the at least one detection objective 90, including the dimensions of the illumination light sheet, may determine the dimensions of the cavities 30 in the third direction A3. The dimensions of the cavities 30 may refer to the dimensions of a single cavity of the cavities 30 or the dimensions of several cavities of the cavities 30. This embodiment enables efficient imaging of multiple samples 50, enabling high-throughput imaging. The plurality of cavities 30 may be spaced apart along the first direction A1 and / or the third direction A3. The plurality of cavities 30 are disposed on the surface 25a of the hydrogel body 25.

[0074] FIG. 3b shows a top view of a sample mount 10 according to another embodiment of the present disclosure. In this embodiment, the sample mount 10 differs from the sample mount 10 shown in FIG. 3a in that each of the multiple cavities 30 is fluidly connected by a channel 31. The channel 31 is formed in the hydrogel 25. The channel 31 may be disposed on the surface 25a of the hydrogel body 25. The channel 31 may have an opening (not shown) for supplying a culture medium to the channel 31. The opening of the channel 31 may be provided on the surface 25a. The fluid connection between the multiple cavities 30 provided by the channel 31 enables biochemical signaling (information exchange) between the multiple samples 50 held in the multiple cavities 30.

[0075] In one embodiment, the microfluidic unit may include a network of channels 31, such as microchannels. The network of channels 31 may be provided in the hydrogel body 25. The network of channels 31 provided in the hydrogel may be a network of microchannels, sometimes called "microvasculature." The network of channels 31 can fluidly connect multiple cavities 30. This fluid connection allows biochemical signaling (information exchange) between multiple samples 50 held in the multiple cavities 30.

[0076] FIG. 4a shows a side view of an objective lens unit 80a of a microscope 80 for imaging at least one sample 50 according to one embodiment of the present disclosure (indicated by a box 80 drawn around the objective lens unit 80a in FIGS. 4a-4c, 5a-5b, and 7). The microscope 80 may be, for example, a light sheet fluorescence microscope. A sample mount 10 may be disposed on the microscope 80. The microscope 80 includes at least one illumination objective lens 85, at least one detection objective lens 90, and the sample mount 10. In the embodiment shown in FIG. 4a, the microscope 80 includes two illumination objective lenses 85 and one detection objective lens 90.

[0077] The at least one illumination objective 85 has an illumination optical axis IA. In one aspect of the present disclosure, the illumination lens 85 can be used more generally to illuminate the at least one sample 50. The at least one detection objective 90 has a detection optical axis DA. The detection optical axis DA is perpendicular to the illumination optical axis IA.

[0078] In the embodiment shown in Figure 4a, the two illumination objective lenses 85 each have an illumination optical axis IA. In the embodiment shown in Figure 4a, the two illumination optical axes IA of the two illumination objective lenses 85 may coincide.

[0079] The detection optical axis DA and the illumination optical axis IA form a plane. The plane formed by the detection optical axis DA and the illumination optical axis IA is, for example, parallel to the plane formed by the second direction A2 and the third direction A3. The plane formed by the detection optical axis DA and the illumination optical axis IA may be disposed perpendicular to the plane.

[0080] The sample holder 10 may include multiple cavities 30 arranged along at least one sampling direction SD. In the embodiment of the present disclosure shown in FIG. 4a, the at least one sampling direction SD is parallel to the first direction A1. In a further embodiment of the present disclosure, the multiple cavities 30 are movable along at least one sampling direction SD. The movement of the multiple cavities 30 allows imaging of multiple samples 50 disposed in the multiple cavities 30. When imaging the samples 50 disposed in the cavities 30, the sample mount 10 is moved in at least one sampling direction SD.

[0081] In the embodiment shown in Figure 4a, the sample mount 10 can be moved in at least one sampling direction SD to image the sample 50. By placing the sample mount 10 in the microscope 80, it is possible to image multiple samples 50, for example, in sequential order. This embodiment of the invention allows multiple samples 50 to be arranged in the cavity 30 along at least one sampling direction SD. Arranging the objective lens unit 80a and the sample mount 10 in this manner may allow for high-throughput imaging of multiple samples 50.

[0082] In a further embodiment, the at least one sampling direction SD may be parallel to both the first direction A1 and the third direction A3. In this embodiment, the sample mount 10 may be movable along both the first direction A1 and the third direction A3. This embodiment enables high-throughput imaging of the at least one sample 50.

[0083] 4b shows a side view of an objective lens unit 80a of a microscope 80 for imaging at least one sample 50 according to a further embodiment of the present disclosure. In the embodiment shown in FIG. 4b, the microscope 80 includes one illumination objective lens 85 and two detection objective lenses 90. The illumination objective lens 85 has an illumination optical axis IA. The two detection objective lenses 90 each have a detection optical axis DA. The two detection optical axes DA may be perpendicular to the illumination optical axis IA of the illumination objective lens 85.

[0084] In the embodiment shown in Figure 4b, the two detection optical axes DA and the one illumination optical axis IA form a plane. In the embodiment shown in Figure 4b, the plane formed by the two detection optical axes DA and the one illumination optical axis IA is parallel to the plane formed by the second direction A2 and the third direction A3. The plane formed by the two detection optical axes DA and the one illumination optical axis IA may also be disposed perpendicularly.

[0085] Similar to the embodiment shown in Figure 4a, the arrangement of objective lens unit 80a and sample mount 10 shown in Figure 4b allows imaging of multiple samples 50 placed in multiple cavities 30 of sample holder 10 by moving sample holder 10 along at least one sampling direction SD. Imaging multiple samples 50 held in multiple cavities 30 allows high-throughput imaging of live samples.

[0086] In the embodiment shown in Figure 4b, the sample mount 10 can be moved in at least one sampling direction SD to allow imaging of multiple samples 50 held in multiple cavities.

[0087] FIG. 4c shows a side view of an objective lens unit 80a of a microscope 80 for imaging at least one sample 50 according to a further embodiment of the present disclosure. In the embodiment shown in FIG. 4c, the microscope 80 includes one illumination objective lens 85 and one detection objective lens 90. The illumination objective lens 85 has an illumination optical axis IA. The detection objective lens 90 has a detection optical axis DA. The detection optical axis DA is disposed at an angle θ relative to the illumination optical axis IA. In one embodiment, the angle θ may be equal to 90° (ninety degrees). In another embodiment, the angle θ may deviate from 90°, depending, for example, on the type of microscope 80 used. In the example shown in FIG. 4c, the cavity axis CA of the at least one cavity 30 is disposed parallel to the illumination objective lens 85 having the illumination optical axis IA and perpendicular to the detection objective lens 90 having the detection optical axis DA. In another embodiment, the cavity axis CA may be perpendicular to the illumination objective 85 having an illumination optical axis IA and parallel to the detection objective 90 having a detection optical axis DA.

[0088] Similar to the embodiment shown in Figures 4a and 4b, the detection optical axis DA and the illumination optical axis IA form perpendicularly arranged planes.

[0089] Similar to the embodiment shown in FIGS. 4a and 4b, the arrangement of the objective lens unit 80a and sample mount 10 shown in FIG. 4c allows imaging of multiple samples 50 placed in multiple cavities 30 of the sample holder 10 by moving the sample holder 10 along at least one sampling direction SD. Imaging multiple samples 50 held in multiple cavities 30 enables high-throughput imaging of live samples. Moving the sample mount 10 in the microscope 80 allows for imaging of numerous samples 50, e.g., in sequential order. Holding multiple samples 50 in multiple cavities of the culture environment 60 allows for cultivating and growing multiple live samples 50. Cultivating and growing multiple samples 50 in multiple cavities 30 allows for imaging of multiple samples 50 at various stages of development, such as embryonic or cell division, or in pathological states, such as cancerous cells. The arrangement shown in Figures 4a-4c allows for high-throughput imaging of multiple samples 50 in developmental or pathological phases.

[0090] 4d and 4e show side views of an objective lens unit 80a of a microscope 80 for imaging at least one sample 50 according to a further embodiment of the present disclosure. A sample mount 10 is also shown in FIGS. 4d and 4e. FIGS. 4d and 4e show an embodiment of the objective lens unit 80a in which at least one illumination objective lens 85 and at least one detection objective lens 90 form a single objective lens 85, 90. The illumination optical axis IA and the detection optical axis DA are disposed at an angle θ relative to each other. The illustrated embodiment enables light sheet microscopy in which the illumination optical axis IA and the detection optical axis DA are disposed non-perpendicular to each other. The multiple cavities 30 are movable in several sampling directions SD. For example, the multiple cavities 30 are movable in three sampling directions SD1, SD2, and SD3, as shown in FIGS. 4d and 4e.

[0091] For example, a single objective lens 85, 90 can generate a light sheet for illuminating at least one sample 50, the light sheet being positioned non-perpendicular to the detection optical axis DA (tilted plane microscopy). The disclosure of EP 2316048 B1 regarding tilted plane microscopy is hereby incorporated in its entirety. The disclosure of WO 2018 / 033581 B1 regarding tilted plane microscopy is hereby incorporated in its entirety. The disclosure of U.S. Patent Application Publication No. 2024 / 0045195 B1 regarding tilted plane microscopy is hereby incorporated in its entirety. The disclosure of EP 3095001 B1 regarding tilted plane microscopy is hereby incorporated in its entirety. The disclosure of U.S. Patent No. 11243391 B1 regarding tilted plane microscopy is hereby incorporated in its entirety.

[0092] The use of a single objective lens 85, 90 in the objective lens unit 80a of the microscope 80 allows for greater freedom of movement of the specimen mount 10. The increased freedom of movement of the specimen mount 10 allows for increased throughput of the imaging method according to the present disclosure.

[0093] Figures 4d and 4e show further embodiments of the sample mount 10. In Figure 4d, the hydrogel body 25 of the sample mount 10 has a substantially triangular cross-section. In Figure 4e, the hydrogel body 25 of the sample mount 10 has a substantially rectangular cross-section.

[0094] FIG. 5a shows a top view of a microscope 80 for imaging multiple samples 50 according to one embodiment of the present disclosure, including multiple sample mounts 10, 10′ arranged in a sample mount stack 10S (see also FIG. 5b). In this embodiment, the sample mounts 10, 10′ may have a cylindrical shape. However, the shape is not limited to a cylindrical shape. The sample mounts 10, 10′ of this embodiment include a hydrogel body 25 and at least one cavity 30 disposed in the hydrogel body 25.

[0095] In this embodiment, the hydrogel body 25 of the sample mount 10, 10' may have a cylindrical shape extending along the second direction A2. The cylindrical block has a radius measured along the first direction A1 and / or the third direction A3. The cylindrical block has a height measured along the second direction A2. The cylindrical block has a central axis extending parallel to the second direction A2.

[0096] The sample mounts 10 and 10' shown in Figure 5b each include at least one cavity 30 and 30'. The at least one cavity 30 and 30' is disposed in the surface 25a and 25a' of the hydrogel body 25 and 25', respectively. The at least one cavity 30 and 30' may be disposed to coincide with the central axis of the cylindrical block of hydrogel 25.

[0097] The specimen mount 10 can move relative to the plane formed by the detection optical axis DA and the illumination optical axis IA of the microscope 80. In the embodiment shown in FIG. 5a, one illumination objective 85 and one detection objective 90 are shown. However, in this embodiment, two illumination objectives 85 and two detection objectives 90 are possible, with the two illumination objectives 85 positioned opposite each other and the two detection objectives 90 positioned opposite each other. The optical axes IA of the two oppositely positioned illumination objectives 85 may coincide. The optical axes DA of the two oppositely positioned detection objectives 90 may coincide. This arrangement enables multi-perspective imaging of multiple specimens 50. Multi-perspective imaging of multiple specimens 50 enables high-resolution imaging and reduces photodamage to the specimens 50. The plane formed by the detection optical axis DA and the illumination optical axis IA is, for example, parallel to the plane formed by the first direction A1 and the third direction A3. The sample mount 10 moves along at least one sampling direction SD. Additionally, the sample mount 10 can rotate about the sampling direction SD to facilitate acquiring multiple images of multiple samples 50 from several angles. This multi-view imaging allows for a larger imaging volume.

[0098] At least one sampling direction SD may extend vertically, at least one sampling direction SD may be parallel to the second direction A2, or at least one sampling direction SD may be parallel to the central axis of the cylindrical block of hydrogel 25.

[0099] The embodiment shown in FIG. 5 a allows for imaging of multiple samples 50 by moving the sample mount 10 in at least one sampling direction SD and / or rotating the sample mount 10 about at least one sampling direction SD. Placing the sample mount 10 in a microscope 80 allows for high-throughput imaging of multiple samples 50. Multiple samples 50 can be imaged in sequential order. This embodiment of the invention allows for multiple samples 50 to be placed in multiple cavities 30. This arrangement may allow for high-throughput imaging of a large number of samples 50 while culturing the multiple samples 50 in a culture environment, as described above.

[0100] FIG. 5b shows a cross-sectional view of a sample mount stack 10S according to one embodiment of the present disclosure. As can be seen from FIG. 5b, multiple sample mounts 10, 10' are stacked on top of each other using at least one connector 35. In one embodiment, the sample mounts 10, 10' may be substantially identical. In one embodiment, the multiple cavities 30 arranged in the sample mount stack 10S may be substantially identical. In another embodiment, different sample mounts 10, 10' may include different numbers of cavities 30 disposed within the hydrogel 25. In a further embodiment, the sample mounts 10, 10' may include cavities 30 having different shapes and / or sizes. In this embodiment, at least one sampling direction SD may be aligned with the cavity axes CD of the multiple cavities 30. The sample mounts 10, 10' are movable along at least one sampling direction SD and rotatable about an axis parallel to the sample direction SD (or A2) to image the sample 50.

[0101] The connector 35 is configured to arrange the plurality of sample mounts 10, 10′ in the sample mount stack 10S. In one embodiment, the connector 35 may be configured to fluidly connect the sample mounts 10 to the perfusion unit 40 via tubing (e.g., microtubing PX as shown in FIG. 5b). The connector 35 may fluidly connect some of the plurality of cavities 30 of the sample mount stack 10S to the perfusion unit 40.

[0102] Arranging the specimen mount stack 10S in the microscope 80 allows multiple specimens 50 to be imaged, e.g., in serial order. This aspect of the invention allows multiple specimens 50 to be placed in multiple cavities 30 of the specimen mount 10, 10'. Such an arrangement allows for high-throughput imaging of multiple specimens 50.

[0103] Figure 6 shows a perspective view of an arrangement 15 including a sample mount 10 according to a further embodiment. The arrangement 15 shown in Figure 6 extends in a second direction A2. The arrangement 15 shown in Figure 6 includes a cap 10a, an outer housing 20a, an inner housing 20b, a hydrogel body 25, a connector 35, and perfusion tubes P1 and P2. The perfusion tubes P1 and P2 may include microtubes.

[0104] The outer housing 20a is connected to the cap 10a of the arrangement 15. The outer housing 20a and the inner housing 20b are transparent. The outer housing 20a forms an outer compartment. The inner housing 20b forms an inner compartment. The inner housing 20b has a smaller diameter than the outer housing 20a. The inner housing 20b is disposed within the outer housing 20a. The inner housing 20b is provided in the outer compartment of the outer housing 20a. The inner housing 20b may be disposed concentrically with the outer housing 20a.

[0105] The outer housing 20a and the inner housing 20b are transparent. The sample mount 10 is disposed within an inner compartment formed by the inner housing 20b. The sample mount 10 includes a hydrogel body 25 and at least one cavity 30. At least one sample 50 is disposed in the at least one cavity 30. In a further embodiment, the sample mount stack 10S may be disposed within the inner compartment formed by the inner housing 20b.

[0106] The inner compartment formed by the inner housing 20b is fluidly connected to the outer compartment formed by the outer housing 20a of the specimen mount 10. In the example shown in Figure 6, the perfusion tubes P1, P2 include a first perfusion tube P1 and a second perfusion tube P2. The first perfusion tube P1 and the second perfusion tube P2 are fluidly connected to a medium exchange circuit or perfusion unit 40 (see also the description of Figure 8).

[0107] In one embodiment, the inner housing 20b may be connected to another inner housing 20b by a connector 35, thereby forming a stack 10S. In another embodiment, the stack 10S may be formed by the outer housing 20a and the connector 35 without using an inner housing 20b. In the embodiment shown in FIG. 6, the sample 50 may be imaged by moving the sample mount 10 in at least one sampling direction SD. Placing the sample mount 10 in a microscope 80 allows multiple samples 50 to be imaged. Imaging may be performed, for example, in sequential order. This arrangement may allow for high-throughput imaging of multiple samples 50.

[0108] Figure 7 shows a perspective view of an embodiment of a microscope 80 according to a further embodiment of the present disclosure. In the example shown in Figure 7, the microscope 80 is arranged with two of the sample mounts 10 shown in Figure 6. However, the number of sample mounts 10 arranged in the microscope shown in Figure 7 may be fewer, i.e., one, or more, i.e., three or more.

[0109] 7, microscope 80 includes two illumination objectives 85 and two detection objectives 90. Each of the two illumination objectives 85 has an illumination optical axis IA. Each of the two detection objectives 90 has a detection optical axis DA. The detection optical axis DA is perpendicular to the illumination optical axis IA.

[0110] The detection optical axis DA and the illumination optical axis IA form a plane. The plane formed by the detection optical axis DA and the illumination optical axis IA is, for example, parallel to the plane formed by the first direction A1 and the third direction A3. The plane formed by the detection optical axis DA and the illumination optical axis IA is, for example, horizontal.

[0111] The microscope 80 further includes a microscope chamber 81, a chamber lid 82, and a specimen holder adapter 83. The microscope chamber 81 is optically accessible to objectives, for example, through openings 84, 86, thereby enabling imaging of at least one specimen 50 mounted on a specimen mount 10 of the microscope chamber 81 using at least one illumination objective 85 and at least one detection objective 90.

[0112] In one embodiment, the microscope chamber 81 includes at least one first lens mount opening 84 for accommodating at least one illumination objective 85 in the at least one first lens mount opening 84 of the microscope chamber 81. The microscope chamber 81 further includes at least one second lens mount opening 86 for accommodating at least one detection objective 90 in the at least one second lens mount opening 86 of the microscope chamber 81. The at least one first lens mount opening 84 and the at least one second lens mount opening 86 allow for installation of the at least one illumination objective 85 and the at least one detection objective 90 in the microscope chamber 81, respectively.

[0113] The at least one first lens mount opening 84 and the at least one second lens mount opening 86 may be sealed using rubber and / or silicone grease to prevent leakage of an immersion medium for immersing the at least one illumination objective 85 and / or the at least one detection objective 90 contained in the sample chamber 81.

[0114] Additionally, the microscope chamber 81 has a bottom opening 88 through which a sample holder adapter 83 is used to access the interior of the microscope chamber 81. The sample holder adapter 83 can be rotated and translated by a sample stage (not shown) to which the sample holder adapter 83 is attached. The sample stage is located below the chamber.

[0115] The top opening 89 is used to install the sample mount 10 into the sample holder adapter 83. Installing the sample mount 10 allows for precise positioning of the at least one sample 50. This aspect further allows for imaging of a region or volume of interest on the at least one sample 50.

[0116] The microscope chamber 81 can be sealed using a chamber lid 82 after the specimen mount 10 is placed in the specimen holder adapter 83. Sealing the microscope chamber 81 prevents unwanted leakage of gas from the microscope chamber 81.

[0117] The chamber lid 82 may have multiple holes for exchanging gases and media using the perfusion tubes P1, P2. In the embodiment shown in FIG. 7, the chamber lid 82 holds adapter pieces for connecting the perfusion tubes P1, P2 from outside the microscope chamber 81 to tubing connected to the cap of the sample holder 10. In another embodiment, the lid 82 may be an integral part of the microscope chamber 81. In another embodiment, multiple holes for exchanging gases and media using the perfusion tubes P1, P2 may be provided in the microscope chamber 81 or in both the microscope chamber 81 and the chamber lid 82.

[0118] The arrangement shown in FIG. 7 would allow for illumination of at least one sample 50 mounted on the sample mount 10 by installing two opposing illumination objectives 85 and two opposing detection objectives 90 in the microscope chamber 81. Furthermore, this arrangement allows for reduced absorption and scattering artifacts when imaging the at least one sample 50, enabling a more homogeneously sampled 3D volume. Furthermore, rotating the at least one sample 50 allows for the sample 50 to be oriented in a desired direction. Furthermore, acquiring a volume from two or more angles (e.g., four or more viewpoints) can increase sampling and approach isotropic resolution.

[0119] FIG. 8 shows a schematic diagram of a medium exchange unit or perfusion unit 40. The medium exchange unit 40 includes a medium exchange circuit. The medium exchange circuit may include at least a first pump PM1, a gas mixer GM, and a microtube PX including at least one tube P1, P2. Depending on the type of the first pump PM1, the medium exchange unit may also include a second pump PM2. In the example shown in FIG. 8, the medium exchange unit 40 is fluidly connected to the sample holder 10 using a first tube P1 and a second tube P2. The medium exchange unit 40 serves to provide the sample holder 10 and / or at least one cavity 30 with culture medium 62 and / or gas 64.

[0120] FIG. 9 shows a flowchart describing a method 100 for manufacturing a sample mount 10. The method 100 includes step S100, which involves providing, e.g., casting, a hydrogel into a mold 20. The hydrogel provided in 20 is moldable. The hydrogel may be a liquid hydrogel. The hydrogel may be made from ground hydrogel particles that are miscible with water. Mixing the ground hydrogel particles with water produces the hydrogel. The mold 20 may be permeable.

[0121] The method further includes step S110 of placing a mold 28 (see FIG. 10 ) within the moldable hydrogel to form at least one cavity 30. The mold 28 may be made of polydimethylsiloxane (PDMS). The mold 28 and the hydrogel body 25 may be covered with phosphate-buffered saline (PBS). The mold 28 may include a plurality of micropillars 29. The micropillars 29 may have a geometric shape corresponding to the geometric shape of the cavities 30. In FIG. 10 , the micropillars 29 are arranged in a two-dimensional array. However, the micropillars 29 may be arranged differently, for example, along a single line or multiple lines. Arranging the micropillars 29 along a line enables the formation of a plurality of cavities 30 arranged along at least one sampling direction SD (see above).

[0122] The dimensions shown in Figure 10 for one of the plurality of micropillars 29 are merely exemplary. In a particular application, the dimensions of the plurality of micropillars 29 can be selected depending on the type of sample 50 to be imaged. Sample types include, but are not limited to, animal or human embryos, tissue samples derived, for example, from biopsies, or organoids.

[0123] The method further includes step S120 of curing the hydrogel to form the hydrogel body 25. Curing of the hydrogel is achieved by allowing the hydrogel to solidify in an incubator. Solidification may take, for example, but is not limited to, 30-40 minutes. The incubator may provide a temperature of 37°C during solidification.

[0124] The method further includes the step S130 of removing the mold 28 from the hardened hydrogel. PBS may be provided prior to the step of removing the mold 28.

[0125] The method may further include a step S140 of treating the surface of the wall 70 of the at least one cavity 30 and / or the surface of the hydrogel body 25. Treating the surface of the wall 70 of the at least one cavity 30 and the hydrogel 25 includes manipulating the physicochemical properties of the hydrogel by light exposure and / or chemical exposure. The physicochemical properties of the wall 70 and / or the hydrogel 25 may be manipulated by at least one of enzymatic degradation, non-enzymatic reaction, UV irradiation treatment, IR irradiation treatment, and / or laser treatment (see above). The laser treatment may include using pulsed laser light having a pulse length in the picosecond or femtosecond range. Absorption of the laser light, particularly nonlinear absorption, allows the laser treatment to be limited to a focal region of the optics used to provide the laser treatment without affecting surrounding regions of the wall 70 and / or the hydrogel 25.

[0126] In one embodiment of the present disclosure, step S140 of treating the surface of the wall 70 of the at least one cavity 30 and / or the surface of the hydrogel body 25 may be performed after the specimen mount 10 is installed in the microscope 80. For example, the treatment of the surface of the wall 70 of the at least one cavity 30 and / or the surface of the hydrogel body 25 may be performed in parallel with a method for imaging the at least one specimen (see below).

[0127] 11 shows a flowchart of a method 200 for imaging at least one sample 50 in a microscope 80. The method 200 includes a step S200 of providing the at least one sample 50 in at least one cavity 30 of the sample mount 10. The step S200 may include accessing the at least one cavity 30 from above and placing the at least one sample 50 into the at least one cavity 30 from above.

[0128] The method further includes a step S210 of culturing at least one sample 50. The culturing step S210 may include providing a culture environment 60 to the at least one cavity 30. Step S210 may include dispensing a culture medium 62 into the at least one cavity 30. Step S210 may further include providing at least one gas, e.g., one or more of oxygen, nitrogen, or carbon dioxide, to the culture environment 60. Step S210 may further include exchanging the culture medium 62 and one or more of the at least one gas 64. In another embodiment, step S210 may be performed before step S200. In a further embodiment, steps S200 and S210 may be performed simultaneously.

[0129] The method for imaging the at least one sample 50 may further include the step S220 of illuminating the at least one sample 50 with at least one illumination objective 85 having an illumination optical axis IA. The method for imaging the at least one sample 50 may further include the step S230 of detecting light emitted or reflected by the at least one sample 50 using at least one detection objective 90 having a detection optical axis DA that is perpendicular to the illumination optical axis IA.

[0130] The method for imaging the at least one sample 50 may further include, in step S240, acquiring at least one image of the at least one sample 50. The at least one image may be acquired in at least one color channel. Acquiring the at least one image may include processing light emitted, transmitted, refracted, scattered, or reflected by the at least one sample 50, for example, to obtain a 2D image and / or a 3D image of the at least one sample 50. In another embodiment, acquiring the at least one image may further include detecting a photoacoustic signal for photoacoustic imaging.

[0131] The method 200 for imaging at least one sample 50 may further include a step S250 of moving the sample mount 10 in at least one sampling direction SD. Step S250 may further include rotating the at least one sample 50, for example, about the at least one sampling direction SD. Moving and / or rotating the sample mount 10 allows for sampling of a three-dimensional sample volume, for example, an image stack.

[0132] The method 200 for imaging at least one sample 50 may return to step 220 of illuminating the at least one sample 50 after step S250 of moving the sample mount 10 in at least one sampling direction SD.

[0133] The method 200 for imaging a sample 50 allows for imaging of the at least one sample 50 while culturing the at least one sample 50 in a culture environment. Culturing allows for growth and / or nutrition of the at least one sample 50, thereby allowing for imaging of the at least one sample 50 over a period of time, e.g., a developmental stage, during which the at least one sample 50 can develop and / or grow. Thus, the imaging method allows for imaging of the at least one sample 50 while the at least one sample 50 is undergoing developmental changes. Moving the at least one sample 50 along at least one sampling direction SD allows for high-throughput imaging of the at least one sample 50.

[0134] This culture further allows for manipulation of growth and / or development conditions during the experiment.

[0135] The microscope 80 may further include a control system for controlling the culture environment (e.g., gas and / or culture medium) and / or the light irradiation treatment (e.g., UV irradiation treatment and / or IR irradiation treatment). The control may be based on imaging of the at least one sample. The imaging allows for the measurement of parameters related to the growth and / or development of the at least one sample 50. The measured parameters can be used to change the control settings of the control system. In this way, feedback from the detected growth and / or development of the at least one sample 50 to the culture of the at least one sample 50 is achieved.

[0136] Reference number 10. Sample Mounting 10S specimen mount stack 20 Housing, type 25 Hydrogel body 28 Press mold 29 Micropillar 30 cavities 30a First Section 30b Second Section 35 Connector PX Microtube P1 First Irrigation Tubing Connector / Adapter P2 Second Irrigation Tubing Connector / Adapter 40 Medium Exchange Unit PM1 First Pump PM2 Second Pump GM Gas Mixer 50 samples 60 Culture environment 62 Culture Medium 64 Gas 66 Discarded medium 70 Wall 80 Microscope 80a Objective Lens Unit 81 Microscope Chamber 82 Microscope Lid 83 Sample holder adapter 84 First lens mount opening 85 illumination objective lens 86 Second lens mount opening 88 Bottom opening 89 Upper opening 90 detection objective lens IA Lighting optical axis DA detection optical axis SD Sampling Direction A1 First Direction A2 Second Direction A3 The third direction

Claims

1. A sample mount (10) for imaging at least one sample (50), comprising: a hydrogel body (25), and at least one cavity (30) arranged in said hydrogel body (25) for holding said at least one sample (50) in a culture environment (60), said at least one cavity (30) having an opening (25b) that provides access to said at least one cavity (30) for placing said at least one sample (50) in said at least one cavity (25) and for providing culture medium and / or gas to said at least one sample (50); A sample mount (10) comprising:

2. 2. The specimen mount (10) of claim 1, wherein the culture environment (60) comprises a gas, such as oxygen, nitrogen, and / or carbon dioxide, and / or a liquid culture medium.

3. 3. The sample mount (10) of claim 1 or 2, wherein the at least one cavity (30) is dimensioned to provide a matrix for providing mechanical support for positioning, growing, and / or developing the at least one sample (50).

4. The sample mount (10) according to any one of claims 1 to 3, wherein the sample mount (10) includes a plurality of cavities (30), and the plurality of cavities (30) are arranged along a first direction (A1), a second direction (A2), and / or a third direction (A3).

5. The sample mount (10) of any of claims 1 to 4, wherein the hydrogel body (25) comprises one or more hydrogels arranged in one or more layers.

6. 6. The specimen mount (10) of claim 5, wherein the plurality of cavities (30) are arranged in several layers of the one or more layers.

7. The specimen mount (10) according to any one of claims 1 to 6, wherein the hydrogel body (25) is made from animal, plant and / or fungal sources, or from chemically synthesized sources.

8. 8. The sample mount (10) of claim 7, wherein the hydrogel body (25) comprises one or more components, the one or more components comprising one or more amino acids, polypeptides, proteins, nucleotides, oligonucleotides, nucleic acids, carbohydrates, lipids, vitamins, choline, minerals, and / or biological cells.

9. The specimen mount (10) of claim 8, wherein the one or more proteins include one or more of laminin, nidogen, collagen, glycoprotein, and / or proteoglycan.

10. 9. The specimen mount (10) of claim 7 or 8, wherein the one or more carbohydrates comprise agarose and / or polyethylene glycol.

11. 11. The specimen mount (10) of claim 1, wherein the hydrogel body (25) is cross-linked and / or coated with one or more of a peptide, e.g., arginylglycylaspartic acid, and / or extracellular matrix components, e.g., laminin, collagen, proteoglycans, and / or glycoproteins.

12. 12. The specimen mount (10) according to any one of claims 1 to 11, wherein the physicochemical properties of the hydrogel body (25) are manipulated by at least one of enzymatic degradation, non-enzymatic reaction, UV light irradiation treatment, and / or IR light irradiation treatment.

13. A specimen mount (10) according to any one of claims 1 to 12, wherein the at least one cavity (30) extends along a cavity axis (CA), the cavity axis (CA) being parallel to the second direction (A2) or inclined relative to the second direction (A2).

14. A sample mount (10) according to any one of claims 1 to 13, further comprising a connector (35) attachable to the sample mount (10), wherein the sample mount (10) attached to the connector (35) can be arranged in a sample mount stack (10S) including a plurality (10, 10') of the sample mounts (10).

15. 15. The sample mount (10) of claim 14, wherein the connector (35) is configured to fluidly connect the sample mount (10) with the culture medium and / or the gas to provide the culture environment (60).

16. An objective lens unit (81 a) for a microscope (80) for imaging a sample (50), comprising: at least one illumination objective (85) with an illumination optical axis (IA), at least one detection objective (90) having a detection optical axis (DA) perpendicular to said illumination optical axis (IA), and A specimen mount (10) according to any one of claims 1 to 14. An objective lens unit (81a) including:

17. 17. The objective lens unit (81a) of claim 16, wherein the sample mount (10) includes a plurality of cavities (30), the plurality of cavities (30) being movable along at least one sampling direction (SD).

18. 18. The objective lens unit (81a) according to claim 16 or 17, wherein the at least one illumination objective lens (85) and the at least one detection objective lens (85) form a single objective lens (85, 90).

19. A method (100) for manufacturing a specimen mount (10) for a microscope (80), comprising the steps of: - providing a moldable hydrogel in a mould (20) (S100); - placing a mold (28) in said moldable hydrogel (S110); - curing (S120) said hydrogel (25) to form a hydrogel body (25) by means of said mold and to form at least one cavity (30) by means of said die (28); and - Removing the mold (28) from the hardened hydrogel body (25) (S130). A method (100) comprising:

20. 20. The method of claim 19, further comprising treating (S140) a surface of the at least one cavity (30) and / or the body of the hydrogel (25).

21. A method (200) for imaging at least one sample (50) in a microscope (80), comprising: - providing (S200) said at least one sample (50) in at least one cavity (30) of a sample mount (10) according to any one of claims 1 to 14, - providing (S210) a culture environment (60) in said at least one cavity (30); - illuminating (S220) said at least one sample (50) by means of at least one illumination lens (85) having an illumination optical axis (IA); - detecting (S230) light emitted, transmitted, refracted, scattered or reflected by said at least one sample (50) using at least one detection objective (90) having a detection optical axis (DA) perpendicular to said illumination optical axis (IA), and - acquiring (S240) at least one image of said at least one sample (50) in said at least one cavity (30); A method (200) comprising:

22. 22. The method (200) for imaging a specimen (50) of claim 21, further comprising: moving (S250) the specimen mount (10) in at least one sampling direction (SD).

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