Method for manufacturing a microfluidic device for automatic microscopic imaging of bioorganisms

Abstract

The invention relates to a microfluidic device (1) for automatic microscopic imaging of bioorganisms, wherein the device (1) comprises a plurality of chambers (2) configured to receive individual bioorganisms, wherein the microfluidic device (1) comprises an alignment portion (3) arranged at a predetermined position and orientation with respect to the chambers (2), wherein the alignment portion (3) comprises a plurality of markers arranged in a pattern (5), wherein the markers (4) of the pattern (5) are arranged with respect to a central point (6) of the pattern (5) such that for any position of a field of view comprising only a fraction of the pattern (5), an automatic centering procedure of the field of view toward or to the central point (6) of the pattern (5) is derivable. The invention further relates to microscope systems for automatic microscopic imaging of bioorganisms, a method for automatized microscopic imaging of bioorganisms using a microscope system, a computer program as well as a method for manufacturing the microfluidic device (1) for automatic microscopic imaging of bioorganisms.

Description

[0001] Method for manufacturing a microfluidic device for automatic microscopic imaging of bioorganisms

[0002] Description:

[0003] The present invention relates to a method for manufacturing a microfluidic device for automatic microscopic imaging of bioorganisms.

[0004] In the realm of high-throughput biological screening, particularly for applications such as genetic and drug screening, existing methods face significant challenges in both the fabrication of microfluidic devices and the automation of microscopy-based imaging. Current fabrication techniques for polydimethylsiloxane (PDMS) devices often suffer from limitations in reliability, scalability, and throughput, which can impede consistent results and slow down research workflows. Additionally, the automated positioning of microscope systems for image acquisition on microfluidic devices remains a complex and often manual process. This complexity can result in decreased imaging speed, lower throughput, and variability in image quality, especially when dealing with large numbers of samples.

[0005] Currently, available techniques lack a robust and scalable solution that integrates reliable microfluidic device fabrication with fully automated and precise microscope positioning. These limitations hinder the ability to conduct high-throughput screenings efficiently, particularly when working with small model organisms like Caenorhabditis elegans or other biological samples. The need for improved systems that can address these drawbacks is crucial for advancing research in genetics, drug discovery, and other fields that rely on high-throughput imaging and analysis.

[0006] Based on this, it is subject of the present invention to provide a method for manufacturing a microfluidic device for automatic microscopic imaging of bioorganisms that provides solutions to the drawbacks of the prior art outlined above.

[0007] This task is solved by a a method for manufacturing a microfluidic device for automatic microscopic imaging of bioorganisms with the features of claim 1.

[0008] Advantageous embodiments of the invention are given in the corresponding dependent claims and described in the following.

[0009] A first aspect of the invention relates to a microfluidic device for automatic microscopic imaging of bioorganisms, wherein the device comprises a plurality of chambers configured to receive individual bioorganisms, wherein the microfluidic device comprises an alignment portion arranged at a predetermined position and orientation with respect to the chambers, wherein the alignment portion comprises a plurality of markers arranged in a pattern, wherein the markers of the pattern are arranged with respect to a central point of the pattern such that forany position of a field of view comprising only a fraction of the pattern, an automatic centering procedure of the field of view toward or to the central point of the pattern is derivable.

[0010] As such, upon centering the field of view to the central point of the pattern, a target position of the device relative to the central point of the pattern, particularly to a target chamber of the plurality of chambers can be approached, particularly in an automatic centering procedure. For example, the bioorganisms to be imaged using the microfluidic device are or comprise multicellular model systems or small animals, embryos, larvae or cells.

[0011] In an embodiment of the invention, at least some markers of the pattern are arranged symmetrically with respect to the central point of the pattern.

[0012] In another embodiment of the invention, at least some of the markers are arranged in marker sets, wherein markers of different marker sets differ from each other in at least one of the following parameters: their sizes, their shapes, a spacing between the markers.

[0013] In the context of the present invention, a “marker set” can comprise one single marker or a plurality of markers, for example two, three, four, five or more markers. The markers, that is, all markers, associated to a specific marker set are preferably identical.

[0014] According to an embodiment of the invention, said parameters of the markers of different marker sets change with their distance and / or their direction with respect to the central point of the pattern according to a predetermined scheme, such that the automatic centering procedure is derivable from detecting at least one parameter of a marker in the field of view comprising only a fraction of the pattern. The predetermined scheme can encode relative positional information of each marker with respect to the central point of the pattern, enabling orientation within the pattern from the detection of a single marker parameter. For example, according to the predetermined scheme, the markers of different marker sets become bigger as their distance to the central point of the pattern increases. For example, markers of a first marker set comprise a diameter of 50 pm and markers of a second marker set comprise a diameter of 70 pm, wherein the markers of the first marker set are closer to the central point of the pattern than the markers of the second marker set. Hence, the automatic centering procedure can be derived from detecting the size of at least one marker in the field of view using the known, predetermined scheme. In another example, according to the predetermined scheme, the shape of markers of different marker sets change with their distance to the central point in a predetermined sequence, for instance, squared markers are located closer to the central point of the pattern than circular markers. In this case, the automatic centering procedure can be derived from detecting the shape of at least one marker in the field of view using the known, predetermined scheme. This measure allows gaining information on the distance and / or direction of a current field of view comprising one or more markers of a specificmarker set to the central point of the pattern, which simplifies the alignment and orientation of and on the device.

[0015] A first and a second axis can define a coordinate system of the microfluidic device, particularly an orthogonal coordinate system. The central point of the pattern can form the origin of this coordinate system.

[0016] In another embodiment of the invention, at least some of the marker sets are arranged along the first axis, wherein the first axis comprises the central point of the pattern. However, not all markers of a specific marker set need to be arranged on the first axis. For example, a marker set can comprise markers forming corners of a polygon, wherein a central point of the marker set is arranged on the first axis.

[0017] According to an embodiment of the invention, the markers of a first marker set are arranged concentrically around the central point of the pattern.

[0018] In another embodiment of the invention, a second marker set is arranged on each side of the first marker set along the first axis. In other words, one second marker set is arranged on a first side of the first marker set along the first axis and another second marker set is arranged on a second side of the first marker set along the first axis, wherein the second side is opposite to the first side.

[0019] In yet another embodiment of the invention, the first marker set and / or the second marker set consists of four markers arranged such that they form a square, respectively, with two of the four markers being arranged on the first axis and the other two of the four markers being arranged on an axis perpendicular to the first axis.

[0020] According to an embodiment of the invention, the markers of the first marker set are smaller than the markers of the second marker set.

[0021] In another embodiment of the invention, a third marker set is arranged on the first axis next to the second marker set and facing away from the first marker set. For example, the markers of the first marker set are arranged concentrically around the central point of the pattern, and one second marker set and one third marker set are arranged on each of the two sides with respect to the central point of the pattern along the first axis.

[0022] For example, each third marker set consists of a single marker.

[0023] According to an embodiment of the invention, the marker or the markers of the second marker set is or are smaller than the markers or the marker of the third marker set.

[0024] In another embodiment of the invention, the marker or the markers of the first marker set is or are smaller than the marker or the markers of the second marker set and wherein the markeror the markers of the second marker set is or are smaller than the markers of the third marker set.

[0025] According to an embodiment of the invention, the markers of the first marker set comprise a smaller spacing than the markers of the second marker set. The spacing refers to a distance between individual markers of the respective marker set. For a marker set with more than two markers, which as such form corners of a polygon, the spacing can be interpreted as the distance between next neighbor markers. The spacing between markers of a specific marker set can be identical, for example the markers are arranged in a square.

[0026] In yet another embodiment of the invention, the markers of the first marker set, the second marker set and / or the third marker set are circular, polygonal, particularly rectangular, or square.

[0027] According to an embodiment of the invention, at least some marker sets are arranged along the second axis, wherein the second axis extends perpendicular to the first axis and through the central point of the pattern. That is, the pattern can comprise different marker sets that are arranged in a cross-shape along the first and the second axis, wherein the central point forms the center of the cross and the crossing point between the first and the second axis.

[0028] In another embodiment of the invention, the marker sets are arranged in an isotropic manner along the first and the second axis with respect to the central point of the pattern, particularly with respect to the first marker set. For example, the size of the markers decreases in the same manner along both sides of the first axis and the second axis, respectively, with respect to the central point of the pattern and / or the distance between adjacent markers along both sides of the first axis and the second axis, respectively, with respect to the central point of the pattern is identical or changes in the same manner.

[0029] For example, the pattern comprises one first marker set with small markers arranged concentrically around the central point of the pattern, as well as four second marker sets with medium-sized markers, wherein a second marker set is arranged on each of the two sides of the first marker set viewed along both the first and the second axis, as well as four third marker sets with large markers that are each arranged next to an associated second marker set and facing away from the first marker set, such that two of the third marker sets are arranged on the first axis and the other two of the third marker sets are arranged on the second axis. In an embodiment of the invention, the chambers of the microfluidic device comprise a first aspect ratio between their length and width of the device between 1 and 50. The length and the width refer to lateral dimensions of the chambers of the device that can be expressed by a coordinate system spanned, for example, by said first and second axis of the pattern.Particularly, the chambers of the microfluidic device comprise a second aspect ratio between their height and width between 0.1 and 2. The height refers to vertical dimensions of the chambers of the device, expressing their spatial extent perpendicular to said first and second axis.

[0030] In particular, the chambers of the microfluidic device comprise a third aspect ratio between their height and length between 0.1 to 50.

[0031] For example, the chambers of the microfluidic device comprise the following dimensions:

[0032]

[0033] According to another embodiment of the invention, the microfluidic device comprises an edge delimiting the microfluidic device in a plane comprising the chambers and the alignment portion, wherein the edge extends comprises at least one edge section with a defined rotational orientation to the first axis or the second axis. For example, the edge section can extend parallel to the first axis or to the second axis, such that the edge section comprises a defined rotational orientation to the first axis or the second axis. Alternatively, the edge section can extend along an axis enclosing a defined angle to the first axis or the second axis. In particular, the edge extends with a first edge section parallel to the first axis and with a second edge section parallel to the second axis. More particularly, the first and the second section meet at a right angle.

[0034] A second aspect of the invention relates to a microscope system for automatic microscopic imaging of bioorganisms, comprising the microfluidic device according to the first aspect of the invention, as well as an optical microscope configured to record at least one image of the microfluidic device. To this end, the optical microscope is preferably arranged such that its field of view can capture at least portions of the chambers in a top view onto the device. In particular, the microscope system comprises an actuator unit configured to move the optical microscope and the device relative to each other. The field of view, particularly a center of the field of view, can be parametrized in a two-dimensional coordinate system, spanned for example by the central point of the pattern and said first and second axis.

[0035] In the context of the present invention, an optical microscope can be understood as a microscope configured to record images based on illumination at least within the visible range of electromagnetic radiation, i.e. electromagnetic radiation with wavelengths between 380 nm and 780 nm, and optionally including the ultraviolet range, i.e. electromagnetic radiation with wavelengths between 100 nm and 380 nm and / or the infrared range, i.e. electromagneticradiation with wavelengths between 780 nm and 1400 nm. The illumination can be realized for example by means of a light emitting diode. The optical microscope can comprise a camera for recording the images, as well as optical elements, for example a lens, particularly an immersion lens, a beam splitter, an image splitter, an emission filter. In particular, the optical microscope can be realized as a fluorescence microscope.

[0036] Particularly, the microscope system comprises a receiving portion for receiving the microfluidic device, wherein the receiving portion for receiving the microfluidic device is shaped complementary to at least the first edge section of the edge of the microfluidic device, such that a coordinate system of the field of view of the microscope can be oriented according to a coordinate system defined by the first axis and the second axis of the microfluidic device by placing the microfluidic device against the receiving portion of the microscope. Particularly, the receiving portion of the microscope system is shaped complementary to the first edge section and the second edge section of said edge of the microfluidic device. This measure allows aligning the coordinate system of the field of view of the microscope with the alignment portion of the device by means of its first and second axis in the sense that orientations of the orthogonal axes defining the coordinate system of the microscope on the one hand and the alignment portion of the microfluidic device on the other hand, essentially coincide in terms of their respective rotational orientation. Hence, no further alignment procedure is necessary to rotationally align the coordinate system of the microscope with the coordinate system of the microfluidic device, as the rotational alignment is realized by the complementary shapes between the alignment frame of the microfluidic device and the receiving portion of the microscope. The microfluidic device may thus be placed in the receiving portion of the microscope, whereafter said automatic centering procedure and / or the method for automatized microscopic imaging of bioorganisms according to the third aspect of the invention is initiated. This third aspect is described in the following.

[0037] A third aspect of the invention relates to a method for automatized microscopic imaging of bioorganisms using the microfluidic device of the first aspect of the invention or the microscope system according to the second aspect of the invention. The method comprises the steps of:

[0038] a. By means of said optical microscope of the microscope system, recording a first image of at least a portion of the pattern in the field of view of the optical microscope, b. based on the arrangement of the markers of the pattern with respect to the central point of the pattern, identifying from the first image the central point of the pattern or, if the central point of the pattern is not identified, an intermediate point between the central point of the pattern and the central point of the field of view of the optical microscope, c. aligning the field of view of the optical microscope and the central point pattern by moving the optical microscope and the microfluidic device with respect to each other,such that the central point of the field of view of the optical microscope coincides with the central point of the pattern or, if the central point of the pattern is not identified, with the intermediate point.

[0039] For example, for a pattern that comprises a plurality of marker sets, wherein the markers of the different marker sets increase in size with the distance of the respective marker set to the central point, the central point or the intermediate point can be identified from marker sizes of marker sets in the current field of view due to the known arrangement of the markers with respect to the central point. Sizes of markers within the field of view can be determined from a known magnification value of the optical microscope.

[0040] According to an embodiment of the invention, if the central point of the pattern is not identified on the first image, at least one further image is recorded of a portion of the pattern in the field of view around at least one intermediate point or around the first image and the optical microscope and the microfluidic device are moved with respect to each other until the central point of the pattern is identified, particularly until the central point of the field of view of the optical microscope coincides with the central point of the pattern.

[0041] In another embodiment of the invention, if the central point of the pattern is not identified on the first image or at least one further image, a tile image is recorded, wherein the tile comprises a plurality of images depicting portions of the pattern located around the portion of the pattern associated with the first image or at least one further image, and wherein the central point of the pattern or, if the central point of the pattern is not identified, the intermediate point is identified from the tile image.

[0042] In yet another embodiment of the invention, the intermediate point or the central point of the pattern is identified based on markers of the pattern, wherein the markers comprise one of the following shapes: circular, polygonal, particularly rectangular or square.

[0043] In an embodiment of the invention, the intermediate point corresponds to a center point between a plurality of markers. For example, the plurality of markers can be the markers associated with a specific marker set, such that the center point between the plurality of markers corresponds to the center point of a specific marker set. For example, if the marker set consists of four markers arranged in a square-shape, the intermediate point can be a center point of the marker set. This measure simplifies the recognition of the intermediate point as well as the accuracy of the alignment.

[0044] In yet another embodiment of the invention, at least one target chamber of the chambers is selected, and wherein upon aligning the field of view of the optical microscope and the central point of the pattern, the optical microscope and the microfluidic device are moved with respect to each other, such that the field of view of the optical microscope coincides with at least a partof the target chamber, particularly wherein at least one microscopic image of the at least part of the target chamber is recorded. As such, the method allows for an automatized movement of the field of view to a selected chamber as well as an automatized imaging, enabled by the predetermined position and orientation of the central point of the pattern in said alignment portion with respect to the chambers.

[0045] A fourth aspect of the invention relates to a method for manufacturing a microfluidic device for automatic microscopic imaging of bioorganisms, wherein the method comprises the steps of:

[0046] a. On a wafer, depositing a film of curable material,

[0047] b. Generating a first positive master mold of cured curable material forming a first set of raised portions on the wafer, wherein the first set of raised portions define chambers and an alignment portion of the microfluidic device, wherein the alignment portion comprises a plurality of markers arranged in a pattern, c. Preparing a first elastomer negative corresponding to the first positive master mold by casting an elastomer compound on the first positive master mold, such that the first elastomer negative comprises a first set of recesses mating the first set of raised portions of the first positive master mold,

[0048] d. Arranging and aligning the first elastomer negative in a first casting dish, wherein the first casting dish comprises a bottom with a platform that is elevated with respect to the bottom, wherein the platform comprises alignment markers for aligning the first elastomer negative on the platform of the first casting dish, wherein the alignment markers define at least a first axis for the microfluidic device, particularly a rotational orientation of respective extension axes of chambers for receiving bioorganisms and wherein the first casting dish comprises a frame for circumferentially enclosing the bottom of the first casting dish, with the first set of recesses of the first elastomer negative being aligned on the alignment markers of the platform, wherein the frame comprises at least a section with a defined rotational orientation to said first axis,

[0049] e. Casting a first casting compound into a circumferential gap between the frame of the first casting dish and the platform with the first elastomer negative aligned on the platform, to obtain a second casting dish comprising the first elastomer negative and the first casting compound attached circumferentially around the first elastomer negative, wherein the second casting dish comprises at least a portion with a defined rotational orientation to said first axis,

[0050] f. Removing the first casting dish from the second casting dish to expose a main recess of the second casting dish mating the elevation of the platform with respect to the bottom of the first casting dish, wherein at least one wall portion forming the main recess (27a) comprises a defined rotational orientation to said first axis,g. Casting a second casting compound into the main recess of the second casting dish, to obtain a second positive master mold with a second set of raised portions mating the first set of recesses of the second casting dish and corresponding to the first set of raised portions of the first master mold, wherein at least one edge portion of the second positive mater mold comprises a defined rotational orientation to said first axis,

[0051] h. arranging a third casting dish around the second set of raised portions of the second positive master mold, such that the third casting dish circumferentially encloses the second set of raised portions of the second positive master mold, wherein at least one edge portion of the third casting dish comprises a defined rotational orientation to said first axis,

[0052] i. casting the third casting dish with an elastomer, particularly with PDMS, to obtain a first portion of the device being structured on a first side according to the first elastomer negative with a third set of recesses mating first and the second set of raised portions of the first and the second positive master mold, wherein at least one edge portion of the elastomer, particularly of the PDMS, comprises a defined rotational orientation to said first axis,

[0053] j. removing the first portion of the device from the second positive master mold, k. attaching a second portion by means of a transparent cover layer to the first side of the first portion to form the microfluidic device.

[0054] The alignment markers in the context of step d. allow to define an axis for the final microfluidic device that can be used to define a coordinate system for the microfluidic device. This coordinate system can be defined by the first and the second axis as disclosed therein, which comprise a defined rotational orientation with respect to the spatial extent of the chambers in the microfluidic device. As the manufacturing method disclosed above in each steps maintains an orientation of at least an edge portion of the various casting dishes and molds at a defined orientation to said axis, for example parallel to said axis, the final microfluidic device can be rotationally aligned with a receiving portion of the microscope system as disclosed herein that is formed complementary to the at least one edge portion by simply placing the edge portion of the final microfluidic device against the receiving portion. The above method thus allows for a simplified and more efficient automatic microscopic imaging of bioorganisms.

[0055] The third casting dish, which is arranged around the second set of raised portions of the second positive master mold, can be casted such with said elastomer, that a volume laterally enclosed by the third casting dish on a surface of the second positive master mold is filled partially or completely by said elastomer. This “volume” is also referred to as a “cavity” herein, in embodiments where the volume is delimited below by said second positive master mold, laterally by the third casting dish, and from the top via said lid. The elastomer ultimately formsthe first portion of the microfluidic device that is patterned with the chambers of the microfluidic device. The cured elastomer filling said volume can remain permanently connected to the third casting dish, wherein the third casting dish ultimately forms a portion of a frame structure delimiting the microfluidic device. The permanent connection between the elastomer, particularly the PDMS, allows for an alignment between the chambers and the frame structure, enabling downstream automation for experiments using the microfluidic device, particularly based on automated image acquisition.

[0056] For example, the wafer is a silicon wafer, a glass wafer or a plastic wafer.

[0057] The film of curable material may, for example, comprise or consist of a photoresist compound. The first elastomer negative can, for example, comprise or consist of an elastomer, particularly a polyurethan elastomer, PDMS or silicone.

[0058] The elastomer compound casted on the first positive master mold can likewise comprise or consist of an elastomer, particularly a polyurethan elastomer, PDMS or silicone.

[0059] For example, the first casting dish and its components, such as the bottom, the platform and the frame can comprise or consist of plastic or metal, particularly aluminum.

[0060] The first casting compound can, for example, comprise or consist of an elastomer, particularly a polyurethan elastomer or silicone.

[0061] The second casting compound can comprise or consist of resin, particularly polyurethane or epoxy casting resin.

[0062] The third casting dish can comprise or consist of plastic or metal, particularly aluminum. In particular, the third casting dish is filled with an elastomer comprising or consisting of PDMS, such that the first portion of the device comprises or consists of PDMS.

[0063] For example, the cover layer can comprise or consist of glass.

[0064] The first set of raised portions ultimately define the chambers and the alignment portion with the pattern of markers of the microfluidic device.

[0065] Step b. can be executed more than once to prepare a plurality of first positive master molds, wherein each positive master mold comprises a respective first set of raised portions. The following steps of the method of manufacturing can thus be parallelized with multiple first positive master molds, which substantially enhances the output of the method.

[0066] Particularly, in step b., at least some of the raised portions can comprise different heights, i.e. some of the raised portions are raised more than others.In particular, step c. can be executed more than once to prepare a plurality of elastomer negatives. Subsequently, in step d., the individual elastomer negatives can be aligned in a larger first casting dish with a plurality of sets of alignment markers. To this end, each elastomer negative can be arranged on alignment markers associated to the respective elastomer negative. Voids between different elastomer negatives can be filled with additional elastomer material. This measure allows fabricating microfluidic devices with a substantially enlarged surface, which further increases the throughput for automatized microscopic imaging. In particular, between steps h. and i. of the method for manufacturing a microfluidic device, a lid comprising at least one access hole is attached to a top side of the third casting dish, such that the third casting dish is sandwiched between the second positive master mold and the lid. A cavity formed by the second positive master mold, the third casting dish and the lid is filled with said elastomer, particularly PDMS, via the access hole of the lid. More particularly, the third casting dish sandwiched between the second positive master mold and the lid are attached to each other. For example, the attachment can be realized by screwing the second positive master mold, the third casting dish and the lid together by one or more screws extending through the second positive master mold, the third casting dish and the lid. Alternatively, the attachment can be realized for example by means of magnets, by means of clamp-connections and / or by elastic elements, particularly elastic bands.

[0067] The attachment between the first portion and the second portion can be realized by means of plasma bonding, particularly by means of an air plasma or an oxygen plasma.

[0068] The cavity can be filled completely with said elastomer, that is, the volume confined by the second positive master mold, the third casting dish and the lid can be completely filled with said elastomer.

[0069] The elastomer filled into the cavity can be cured such that the elastomer forms a permanent mechanical bond with the second positive master mold, the third casting dish and / or the lid. The term “mating” expresses a positive-negative-relation between corresponding structures. For instance, a raised portion mating a recess can be interpreted such that the raised portion is formed such that it is configured to fill out the recess.

[0070] A fifth aspect of the invention relates to a microscope system comprising an optical microscope and a computer adapted to execute the steps of the method according to the third aspect of the invention.

[0071] For example, the means can comprise or consist of a processor unit or a computer.A sixth aspect of the invention relates to a computer program comprising instructions to cause the microscope system according to the fifth aspect of the invention to execute the method according to the third aspect of the invention.

[0072] Exemplary embodiments are described below in conjunction with the Figures. The Figures are appended to the claims and are accompanied by text explaining individual features of the shown embodiments and aspects of the present invention. Each individual feature shown in the Figures and / or mentioned in the text of the Figures may be incorporated (also in an isolated fashion) into a claim relating to the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect and / or the sixth aspect according to the present invention.

[0073] Figs. 1a-d each show an embodiment of an alignment portion of a microfluidic device according to the invention, wherein different positions of a field of view of a microscope for microscopic imaging of bioorganisms for are aligned on the alignment portion;

[0074] Fig. 2 shows an exemplary alignment portion of a microfluidic device according to another embodiment of the invention;

[0075] Fig. 3 shows an embodiment of a microfluidic device according to the invention with multiple chambers arranged next to an alignment portion;

[0076] Fig. 4 schematically depicts an embodiment of the method for manufacturing a microfluidic device according to the invention;

[0077] Fig. 5 shows an embodiment of a first casting dish used to manufacture a second casting dish, which in turn allows for the fabrication of a first portion of a microfluidic device according to the invention;

[0078] Fig. 6 shows a perspective view of an assembly for manufacturing a first portion of a microfluidic device according to the invention;

[0079] Fig. 7 shows a cross-sectional view through a microfluidic device according to an embodiment of the invention: and

[0080] Fig. 8 shows a top view of a microfluidic device according to an embodiment of the invention next to a receiving portion of a microscope system, wherein an edge delimiting the microfluidic device extends in sections parallel to a first axis and a second axis defining a coordinate system of the alignment portion of the microfluidic device.Figs. 1a-d show an embodiment of an alignment portion 3 of a microfluidic device 1 according to the invention. The alignment portion 3 comprises a plurality of markers 4 that are arranged in a pattern 5. The alignment portion 3 is arranged at a predetermined position and orientation with respect to chambers 2 of the microfluidic device 1 , as depicted for example in Fig. 3. The chambers 2 are configured to receive bioorganisms that can be automatically imaged using a microscope system according to the invention. The arrangement of the markers 4 in the pattern 5 allows aligning the field of view of a microscope of the microscope system toward or to said central point 6 of the pattern 5, whereafter the field of view of the microscope can be moved to target chambers 2 of the microfluidic device 1, particularly in an automatized manner.

[0081] The alignment portion 3 according to the present embodiment comprises three different marker sets 4a, 4b, 4c with markers 4 that are arranged around said central point 6. A first marker set 4a comprises four markers 4 that form a square with the center of the square coinciding with the central point 6 of the pattern 5. A respective second marker set 4b is arranged on each side of the first marker set 4a along a first axis 11 of the microfluidic device 1. A third second marker set 4c is arranged next to the first marker set 4a along a second axis 12 of the microfluidic device 1 which extends perpendicular to the first axis 11 , wherein the first and the second axis 11,12 cross at the central point 6 of the pattern 5. In the present embodiment, the markers 4 of the three marker sets 4a, 4b, 4c each comprise a circular shape. However, the markers 4 of the first marker set 4a are smaller than the markers 4 of the second marker set 4b and the markers 4 of the second marker set 4b are smaller than the markers 4 of the third marker set 4c. As such, the markers 4 forming the different marker sets 4a, 4b, 4c become systematically larger as their distance to the central point 6 of the pattern 5 increases, which allows identifying individual marker sets 4a, 4b, 4c and to align the field of view of the microscope with the central point 6 of the pattern 5, whereafter individual chambers 2 of the microfluidic device 1 can be approached.

[0082] Fig. 1a depicts a field of view of the microscope by means of a first image 41. In this case, the center of the field of view is only shifted by an offset of 300 pm with respect to the central point 6 of the pattern 5. From the sizes of the markers 4 of the first marker set 4a visible in the first image 41, the markers 4 can be identified as forming part of the first marker set 4a, that are arranged concentrically around the central point 6. In this case, the microscope and the microfluidic device 1 can thus be moved with respect to each other upon recording of a single image, namely said first image 41, to compensate the offset between the central point of the field of view of the microscope and the central point 6 of the pattern, such that the central point of the field of view of the microscope coincides with the central point 6 of the pattern 5.

[0083] In Fig. 1b, however, the field of view of the microscope corresponding to the first image 41 does not comprise the central point 6 of the pattern, as can be understood from identifying the sizes of the markers 4 arranged within the first image 41. These markers 4 can be identifiedas markers 4 of the second marker set 4b due to their size. To identify the central point 6 of the pattern 5, the microscope records a 3x3 tile image 50 that comprises, besides the first image 41, further images 42,43,44,45,46,47,48,49 of portions of the pattern 5 in respective fields of views around the first image 41. In the present example, a fourth image 44 of the tile image 50 comprises the first marker set 4a, which allows identifying the central point 6 of the pattern 5 as the center of the markers 4 associated to the first marker set 4a, whereafter the field of view of the microscope can be aligned with the central point 6.

[0084] In Fig. 1c, the field of view of the microscope corresponding to the first recorded image 41 does not comprise the central point 6 of the pattern 5, nor markers 4 of any of the marker sets 4a, 4b, 4c. To identify markers 4 of the marker sets 4a, 4b, 4c, a tile image 50 comprising further images 42,43,44,45,46,47,48,49 of portions of the pattern 5 in respective fields of views around the first image 41 is recorded. A third image 43 of the further images 42,43,44,45,46,47,48,49 in the top-left corner each comprises a marker 4 which can be identified as forming part of one of the second marker sets 4b, while the other images 41 ,42,44,45,46,47,48,49 do not comprise markers 4 used for the alignment. The squared markers visible in these other images 41,42,44,45,46,47,48,49 can be used for adjusting a focal plane of the microscope onto the plane comprising the pattern 5. This information allows the microscope to approach the central point of the third image 43 as an intermediate point between the first image 41 and the central point 6 of the pattern 5, in order to iteratively approach the central point 6. Subsequently, another tile image 50 is recorded, wherein the third image 43 of the first tile image 50 forms the first image 41 of the further tile image. The markers 4 of the first marker set 4a and thus the central point 6 of the pattern 5 are now found in one of the images of the further tile image, which allows aligning the field of view of the microscope with the central point 6 of the pattern 5.

[0085] In Fig. 1d, the field of view of the microscope corresponding to the first recorded image 41 does not comprise the central point 6 of the pattern 5, nor markers 4 of any of the marker sets 4a, 4b, 4c, as in Fig. 1c. However, a fifth image 45 of a tile image 50 around the first image 41 comprises a marker 4 forming one of the third marker sets 4c. This marker 4 forms an intermediate point to which the microscope is moved in order to approach the central point 6. At his location, a further tile image is recorded, such that the fifth image 45 of the first tile image 50 corresponds to the first image 41 of the further tile image. One of the images of the further tile images comprises the first marker set 4a, which allows aligning the field of view of the microscope with the central point 6 of the pattern 5 in an iterative manner.

[0086] As a size reference, in the present embodiment, the dashed circles indicate 500 pm, 750 pm and 1000 pm distance from the central point 6 of the pattern 5.Fig. 2 shows an exemplary alignment portion 3 of a microfluidic device 1 according to another embodiment of the invention. The markers 4 of the present alignment portion 3 are arranged in a pattern 5 with three different marker sets 4a, 4b, 4c. A first marker set 4a comprises four circular markers 4 that are concentrically arranged around the central point 6 of the pattern. A respective second marker set 4b is arranged on each side of the first marker set 4a along a first axis 11 of the microfluidic device. A third second marker set 4b is arranged below the first marker set 4a along a second axis 12 of the microfluidic device 1 which extends perpendicular to the first axis 11, wherein the first and the second axis 11,12 cross at the central point 6 of the pattern 5. A respective third marker set 4c consisting of a single marker 4 is arranged next to the second marker sets 4b on the first axis 11 , facing away from the first marker set 4a. All markers 4 in the present embodiment comprise a circular shape, wherein the diameter of the circles of the markers 4 of the first marker set 4a is 50 pm, smaller than the diameter of the circles of the second marker set 4b (70 pm), which in turn is smaller than the diameter of the circles of the third marker set 4c (90 pm). The distance between the markers 4 of the first marker set 4a indicated in Fig. 2 is 170 pm and the distance between the markers 4 of the second marker set 4b is 200 pm. The respective spacing between the first marker set 4a and the second marker sets 4b is 330 pm, which equals the spacing between the second marker sets 4b and their respective neighboring third marker set 4c. As such, the different marker sets 4a, 4b, 4c can be optically clearly distinguished without occupying too much space on the microfluidic device 1. The first marker set 4a remains visible at magnifications of the microscope up to 63x. If larger magnifications are to be used, the dimensions of the markers 4 can be reduced accordingly. For an automatized image recognition and alignment of the microscope and the central point 6 of the pattern 5, the three marker-diameters of the different marker sets 4a, 4b, 4c as well as the minimum distance between the smallest markers 4, i.e. the first marker set 4a, are used as input calibration parameters.

[0087] Fig. 3 shows an embodiment of a microfluidic device 1 according to the invention, particularly a portion of the microfluidic device 1 by means of a first unit 1a of the microfluidic device 1. This first unit 1a is arranged next to an alignment portion 3 of the microfluidic device 1. The first unit 1a of the device comprises a plurality of chambers 2, in the present embodiment 225 chambers, that are arranged in a 15x15 square-matrix. Besides the first unit 1a, the microfluidic device 1 can comprise further units (not shown in Fig. 3) that can - similar to the chambers 2 - be arranged next to each other in a matrix shape on the microfluidic device 1. For example, the full microfluidic device 1 comprises 36 units that are arranged next to each other in a 6x6 square-matrix. Each chamber 2 of every unit has a well-defined distance and orientation with respect to the alignment portion 3 of the microfluidic device 1, particularly the central point 6 of the pattern 5 of the alignment portion 3 that are shown in more detail in the example embodiments of Fig. 1 or Fig. 2. Hence, individual chambers 2 of the microfluidic device 1,particularly of different units such as the first unit 1a, can be approached with the optical microscope of the microscope system according to the invention upon alignment of the field of view of the microscope on the alignment portion 3 of the microfluidic device 1, particularly as shown in Fig. 1. In the present example, a microfluidic device 1 with 36 units and 225 chambers 2 per unit comprises a total of 8100 chambers 2 that can be imaged using the microscope system according to the invention.

[0088] In the present embodiment, each of the chambers 2 has a size of 315x315 pm. Bioorganisms and liquid can be introduced into the chambers 2 via an inlet 7 of the first unit 1a of the microfluidic device 1, such that living bioorganisms can be imaged by the microscope system. Each unit of the microfluidic device 1 can comprise an associated inlet 7 for supplying the respective unit with liquid and bioorganisms.

[0089] Fig. 4 schematically depicts an embodiment of the method for manufacturing a microfluidic device 1 according to the invention. Individual steps of the method according to the present embodiment are referenced with capital letters in Fig. 4 and the following text.

[0090] First (A,B), a film 21 of curable material, such as a photoresist compound, is deposited on a wafer 20. For example, the wafer is a silicon wafer. A first positive master mold 22 of cured curable material is generated on the wafer 20, forming a first set of raised portions 22a on the wafer 20. This can be done, for example, by photolithography (C,D). The first set of raised portions 22a ultimately define the positions and spatial extent of the chambers 2 as well as the alignment portion 3 with the pattern 5 of markers 4 of the microfluidic device 1. To this end, the individual raised portions 22a forming the set of raised portions 22a are separated from each other on the wafer 20, i.e. not interconnected. Some of the raised portions 22a can define the chambers 2 of the microfluidic device 1 , others can define the markers 4 of its alignment portion 3 arranged in a pattern 5. In Fig. 4, a single raised portion 22a is depicted as an example. For example, for realizing chambers 2 with squared cross-sections of lateral dimensions 315x315 pm and heights below 50 pm, the corresponding raised portions 22a may take on corresponding dimensions on the wafer 20, wherein the height corresponds to an elevation on the wafer 20 and the lateral dimensions denote the spatial extent parallel to the extension plane of the wafer 20.

[0091] Next (E), a first elastomer negative 23 corresponding to the first positive master mold 22 is prepared by casting an elastomer compound 24 on the first positive master mold 22, such that the first elastomer negative 23 comprises a first set of recesses 24a mating the first set of raised portions 22a of the first positive master mold 22. In other words, the first set of recesses 24a of the first elastomer negative 23 form a negative of the first set of raised portions 22a of the first positive master mold 22. For example, the elastomer compound and thus the firstelastomer negative comprises or consists of an elastomer, particularly a polyurethan elastomer, PDMS or silicone.

[0092] Now, the first elastomer negative 23 is removed from the first positive master mold 22 (F) and is arranged and aligned in a first casting dish 25 (G). This first casting dish 25 comprises a bottom 25a with a platform 25b that is elevated with respect to the bottom 25a which are shown in more detail in Fig. 5. The platform 25b comprises alignment markers 25c for aligning the first elastomer negative 23 on the platform 25b of the first casting dish 25. The first casting dish 25 further comprises a frame 25d for circumferentially enclosing the bottom 25a of the first casting dish 25, with the first set of recesses 24a of the first elastomer negative 23 being aligned on the alignment markers 25c of the platform 25b. For example, the first casting dish 25 and its components, such as the bottom 25a, the platform 25b and the frame 25d can comprise or consist of aluminum.

[0093] Subsequently (H), a first casting compound 26, for example silicone, is casted into a circumferential gap that is formed between the frame 25d of the first casting dish 25 and the platform 25b as a consequence of the platform 25b being elevated with respect to the bottom 25a and the space between the platform 25b and the lateral side walls of the frame 25d, cf. also Fig. 5. Thereby, a second casting dish 27 comprising the first elastomer negative 23 and the first casting compound, which is now attached circumferentially around the first elastomer negative 23, is obtained.

[0094] The first casting dish 25 can now be removed from the second casting dish 27 to expose a main recess 27a (I) of the second casting dish 27 that mates the elevation of the platform 25b with respect to the bottom 25a of the first casting dish 25. The first set of recesses 24a is arranged in the main recess 27a of the second casting dish 27. As outlined in more detail below, this first casting dish 25 allows fabricating a second positive master mold 29 that in turn can be used to fabricate microfluidic devices 1 according to the invention that have chambers 2 that are well aligned with respect to their lateral boundaries, which allows aligning the coordinate system of the field of view of a microscope system as disclosed herein with respect to the alignment portion of the microfluidic device 1 and its chambers 2. A fabrication of microfluidic devices 1 based on the first positive master mold 22 instead would likely cause the underlying wafer 20 to break and give rise to microfluidic devices 1 with chambers 2 that are randomly oriented with respect to the lateral boundaries of the microfluidic device 1.

[0095] Hence, a second casting compound 28, for example polyurethane, is cast into the main recess 27a of the second casting dish 27, to obtain a second positive master mold 29 with a second set of raised portions 29a (J). This second set of raised portions 29a mates the first set of recesses 24a of the second casting dish 27 and thus corresponds to the first set of raised portions 22a of the first positive master mold 22. For example, the main recess 27a of thesecond casting dish 27 comprises lateral dimensions of 85mmx35mm, which thus define the dimensions of the second positive master mold 29.

[0096] A third casting dish 30 is arranged around the second set of raised portions 29a of the second positive master mold 29 (K), such that the third casting dish 30 circumferentially encloses the second set of raised portions 29a of the second positive master mold 29. In particular, the third casting dish 30 has a rectangular cross-section as shown in Fig. 4, such that it can be arranged in a corresponding receiving structure of the microscope system, which simplifies the alignment procedure on the microfluidic device 1 using the microscope system.

[0097] Optionally (L), a lid 32 with access holes 32a is attached to the top side of the third casting dish 30, forming an assembly 300 with the third casting dish 30 being sandwiched between the second positive master mold 29 and the lid 30. For example, the lid 32 can comprise or consist of PMMA. A similar assembly 300 comprising the second positive master mold 29, the third casting dish 30 and the lid 30 is shown in Fig. 6.

[0098] Now, the third casting dish 30 is cast with an elastomer, particularly with PDMS 31, to obtain a first portion 100 of the microfluidic device 1, which is structured on a first side according to the first elastomer negative 23 with a third set of recesses 31a that mate the first and the second set of raised portions 22a, 29a of the first and the second positive master mold 22,29, respectively. Particularly, if the lid 32 is used, a cavity 33 formed at an inside of the third casting dish 30 sandwiched between the second positive master mold 29 and the lid 30 can be cast with said elastomer via the access holes 32a.

[0099] The first portion 100 of the microfluidic device 1 is now removed from the second positive master mold 29 (L).

[0100] Subsequently, a second portion 200 by means of a transparent cover layer is attached to the first side of the first portion 100 of the microfluidic device 1. The resulting microfluidic device 1 comprising the first portion 100 and the second portion 200 closing the chambers 2 of the microfluidic device 1 realized by the third set of recesses 31a is shown in Fig. 7.

[0101] The present method thus includes the fabrication of a second positive master mold 29, preferably comprising polyurethane, which corresponds in terms of its shape to a first positive master mold 22 that is formed on said wafer 20, particularly using a photoresist. The second positive master mold 28 is mechanically much more stable than the first positive master mold 22 which is based on a typically fragile wafer 20. The enhanced mechanical stability results from the thicker base of the second positive master mold 29 resulting from the casting of the second casting compound 28 in the main recess 27a of the second casting dish 27, compared to the typically much thinner and more fragile wafer 20. For example, the second positive master mold 28 comprises a thickness of 5 mm, wherein conventional wafers 20 typically comprise thicknesses of well below 1 mm. Moreover, a material like polyurethan ismechanically much more resilient than typical wafer materials. The mechanical stability is particularly important when casting the third casting dish 30 with PDMS, which could cause mechanical damage to the wafer material. At the same time, the second positive master mold 29 accurately reproduces the first set of raised portions 22a of the first positive master mold 22 by means of its second set of raised portions 29a. As such, the second positive master mold 29 provides a copy of the first positive master mold 22 with improved mechanical stability, which allows for repeated manufacturing of microfluidic devices 1.

[0102] Moreover, the present method allows producing microfluidic devices 1 whose chambers 2 -that correspond in terms of their shape to the first set of recesses 24a of the first elastomer negative 23 - have a well-defined orientation with respect to the edges of the microfluidic device 1 that are defined by said third casting dish 30: As shown in Fig. 4 and Fig. 6, the third casting dish 30 has a square- or rectangular cross-section, such that it can be aligned on a mating receiving structure of the microscope system in a straight-forward way. This means that the microfluidic device 1 with its chambers 2 formed in the PDMS 31 needs to be well-aligned with respect to the third casting dish 30 framing the PDMS 31, in order to be aligned with the microscope, particularly the receiving portion of the microscope. The first elastomer negative 23 is formed on the wafer 20 without a well oriented frame. Hence, the position of the first set of recesses 24a of the first elastomer negative 23 with respect to its edge is arbitrary, making the first elastomer negative 23 unsuitable for the final microfluidic device 1 as its chambers 2 would not be well aligned with the respect to the third casting dish 30, if the latter was framing the first elastomer negative 23 as such. To solve this problem, the first elastomer negative 23 is aligned with its first set of recesses 24a on the alignment markers 25c of the platform 25b of the first casting dish 25. If the first elastomer negative 23 is larger than the platform 25b, it can be cut into an appropriate shape such that it fits onto the platform 25b with the alignment markers 25c. Now, the circumferential gap between the aligned first elastomer negative 23 and the frame 25d of the first casting dish 25 is cast with said first casting compound 26 to form said second casting dish 27. In this second casting dish 27, the first set of recesses 24a are advantageously aligned with respect to said main recess 27a. As such, the second master mold 28 formed by the second casting dish 27 is not just mechanically stable, but also allows for manufacturing microfluidic devices 1 with well-defined alignment of their chambers 2 with respect to the lateral frame of the microfluidic device 1 defined by the third casting dish 30. Fig. 5 shows an embodiment of a first casting dish 25 used to manufacture a second casting dish 27, which in turn allows for the fabrication of microfluidic devices 1 according to the invention.

[0103] The first casting dish 25 according to the present embodiment comprises a frame 25d and a bottom 25a with a platform 25b that is elevated with respect to the bottom 25a. The frame 25d comprises a first frame part 25d1 and a second frame part 25d2 that serve as lateral side wallsof the first casting dish 25. A third frame part 25d3 forms the lid of the first casting dish 25. The first and the second frame part 25d1,25d2 are attached to the bottom 25a, for example, by means of a screw-connection. The platform 25b comprises four alignment markers 25c for aligning the first set of recesses 24a of said first elastomer negative 23 on the platform 25b. In this configuration, a circumferential gap forms between the first elastomer negative 23 aligned on the platform 25b and the frame 25d, particularly the first and the second frame part 25d1 ,25d2. Said first casting compound 26, for example silicone, is cast into the circumferential gap and connects with the first elastomer negative 23, to form the second casting dish 27 as shown in Fig. 5. The second casting dish 27 comprises a main recess 27a wherein the first set of recesses 24a are well-oriented with respect to the main recess 27a. This allows for the manufacturing of microfluidic devices 1 with chambers 2 that are well-oriented with respect to their lateral boundaries.

[0104] Fig. 5 further shows said second positive master mold 29 that is formed by casting said second casting compound, for example polyurethane, into the main recess 27a of the second casting dish 27. This second positive master mold 29 can ultimately be used to fabricate microfluidic devices 1 according to the invention.

[0105] Fig. 6 displays an assembly 300 for fabricating a first portion 100 of a microfluidic device 1 using the method according to Fig. 4 and the casting dishes 25,27,29 shown in Fig. 5. From bottom to top, the assembly 300 comprises a plurality of screws 35 forming feet of the assembly 300, wherein the screws 35 extend through the second positive master mold 29 which is formed by a polyurethane layer with said second set of raised portions 29a facing the third casting dish 30. The third casting dish 30 is realized as an aluminum frame that circumferentially encloses the second set of raised portions 29a of the second positive master mold 29. A lid 32 formed by a PMMA layer is attached on top of the aluminum frame realizing the third casting dish 30. The second positive master mold 29, the third casting dish 30 and the lid 32 are screwed to each other by means of said screws 35 that engage with respective nuts 36 arranged on a top side of the lid 32, such that a cavity 33 enclosed by the second positive master mold 29, the third casting dish 30 and the lid 32 can be sealed by screwing the structures together. As an alternative to this screw connection, the second positive master mold 29, the third casting dish 30 and the lid 32 can be attached to each other magnetically, by means of clamp-connections and / or by elastic elements, particularly elastic bands. To cast elastomer material, particularly PDMS, into the otherwise sealed cavity 33, the assembly 300 comprises a plurality of access holes 32a that extend through the lid 32 for establishing a fluid connection between the cavity 33 and the outside of the assembly 300.

[0106] As can further be seen in Fig. 6, the assembly 300, particularly the third casting dish 30 comprises a plurality of support bars 34 extending across the cavity 33 so as to connect opposite sides of the third casting dish 30. The support bars 34 are embedded in the elastomer,so as to hold the elastomer in place even if the elastomer material does not stick to the material of the third casting dish 30, as is the case for example for PDMS as the elastomer material and aluminum as the material of the third casting dish 30. In an alternative embodiment, the third casting dish 30 is devoid of support bars 34.

[0107] After casting the cavity 33 with elastomer, particularly PDMS, and curing at room temperature, a first portion 100 of the microfluidic device 1 comprising the third casting dish 30 with the cured elastomer can be removed from the second positive master mold 29 and the lid 32, which may be reused to fabricate further first portions 100 of the microfluidic device 1.

[0108] Fig. 7 shows a cross-section of an embodiment of the final microfluidic device 1 , after removing the first portion 100 of the microfluidic device 1 fabricated as explained in Figs. 4,5 and 6 with corresponding text and attaching it to a second portion 200 of the microfluidic device 1.

[0109] The cross-section through the microfluidic device 1 shows the first portion 100 laterally delimited by the third casting dish 30 that may be realized by said aluminum frame. Support bars 34 extend across the third casting dish 30 and are embedded in the cured elastomer, particularly PDMS, filling the third casting dish 30. A first side of the first portion 100 of the microfluidic device 1 patterned with said third recesses 31a is attached to a flat surface of a second portion 200 of the microfluidic device 1, such that the third recesses 31a are closed by the second portion 200 to form the individual chambers 2 of the microfluidic device 1. In particular, the first portion 100 and the second portion 200 can be attached to each other by means of plasma bonding. For example, the first portion 100 and the second portion 200 are attached to each other by means of an air plasma or an oxygen plasma, wherein the first portion 100 and the second portion 200 become reactive and bond covalently to each other. The second portion 200 comprises or consists of a transparent material, such that the chambers 2, particularly bioorganisms arranged in the chambers 2, can be imaged by an optical microscope through the second portion 200. The bioorganisms and liquid can be conducted into and out of the individual chambers 2 by access holes 100a extending through the first portion 100 to the third recesses 31a forming the chambers 2.

[0110] The access holes 100a in the first portion 100 can be created by piercing a penetrator, such as a needle, through the first portion 100, particularly through the cured PDMS layer, down to the third recesses 31a that form the chambers 2. Multiple access holes 100a can be produced simultaneously by piercing multiple penetrators through the first portion 100, which significantly simplifies and accelerates the fabrication of the microfluidic devices 1. For example, a punching tool with multiple penetrators, such as needles, can be arranged in a predetermined pattern above the first portion 100 to form the access holes 100a simultaneously. Each penetrator can be configured to move along an axis towards and into the first portion 100, creating the access holes 100a in an accurate and efficient way.U. iA / 0 22

[0111] Fig. 8 shows a top view of a microfluidic device 1 according to an embodiment of the invention next to a receiving portion 60 of a microscope system according to an embodiment of the invention. The microfluidic device 1 comprises an edge 1 b that laterally delimits the microfluidic device 1 in a plane containing the chambers 2 and the alignment portion 3. The edge 1b can be formed by the third casting dish 30, such that the microfluidic device 1 shown here can be obtained from the method of fabrication as described in Figs. 4 to 7. As a result of the fabrication, the upper and the lower section of the edge 1b visible in Fig. 8 extend at a defined rotational orientation to the first axis 11 of the alignment portion 3, while the left and right sections of the edge 1b extend at a defined rotational orientation to the second axis 12 of the alignment portion. In particular, the upper and the lower section of the edge 1b extend parallel to the first axis 11 and the left and right sections of the edge 1 b extend parallel to the second axis 12. The upper and the lower sections meet the left and the right sections at right angles. The receiving portion 60 of the microscope is shaped complementary to the edge 1b of the microfluidic device. Specifically, a first section 60a and a second section 60b of the receiving portion 60 join at a right angle, matching the orthogonal structure of edge 1b. Consequently, when the microfluidic device 1 is placed flush against the receiving portion 60 of the microscope, the coordinate system of the microscope’s field of view, which has a defined orientation relative to its receiving portion 60, aligns with the coordinate system of the microfluidic device 1. This alignment orients the axes of the two coordinate systems relative to each other, potentially up to a translational offset.

[0112] The present embodiment substantially simplifies and speeds up the throughput of microscopic imaging, as the microfluidic devices 1 do not need to be aligned in terms of their orientation with respect to the microscope due to the defined orientation of their edge 1b with respect to the receiving portion 60 of the microscope. The automatic imaging of individual chambers 2 can be initiated upon an alignment of the field of view of the optical microscope with the central point 6 of the alignment portion 3 of the microfluidic device 1.

[0113] The microscope system can further comprise a holding fixture for holding the microfluidic device 1 at a position flush against the receiving portion 60, particularly by pressing the microfluidic device 1 against the receiving portion 60, such that the microfluidic device 1 does not move with respect to the microscope system during the automatic imaging procedure.List of reference signs

[0114] Microfluidic device 1 First unit 1a Edge of the microfluidic device 1b Chamber 2 Alignment portion 3 Marker 4 First marker set 4a Second marker set 4b Third marker set 4c Pattern 5 Central point 6 Inlet 7 First axis 11 Second axis 12 Wafer 20 Film 21 First positive master mold 22 First set of raised portions 22a First elastomer negative 23 Elastomer compound 24 First set of recesses 24a First casting dish 25 Bottom of first casting dish 25a Platform of first casting dish 25b Alignment markers of first casting dish 25c Frame of first casting dish 25d First part of frame 25d1Second part of frame 25d2 Third part of the frame 25d3 First casting compound 26 Second casting dish 27 Main recess 27a Second casting compound 28 Second positive master mold 29 Second set of raised portions 29a Third casting dish 30 PDMS 31 Third set of recesses 31a Lid 32 Access hole of the lid 32a Cavity 33 Support bar 34 Screw 35 Nut 36 First image 41 Second image 42 Third image 43 Fourth image 44 Fifth image 45 Sixth image 46 Seventh image 47 Eighth image 48 Ninth image 49 Tile image 50 Receiving portion of the microscope 60First portion of the device 100 Access hole of the first portion 100a Second portion of the device 200 Assembly 300

Claims

u: / O 26Patent claims:

1. A method for manufacturing a microfluidic device (1) for automatic microscopic imaging of bioorganisms, wherein the method comprises the steps of:a. On a wafer (20), depositing a film (21) of curable material,b. Generating a first positive master mold (22) of cured curable material forming a first set of raised portions (22a) on the wafer (20), wherein the first set of raised portions (22a) define chambers (2) and an alignment portion (3) of the microfluidic device (1), wherein the alignment portion (3) comprises a plurality of markers (4) arranged in a pattern (5),c. Preparing a first elastomer negative (23) corresponding to the first positive master mold (22) by casting an elastomer compound (24) on the first positive master mold (22), such that the first elastomer negative (23) comprises a first set of recesses mating the first set of raised portions (22a) of the first positive master mold (22),d. Arranging and aligning the first elastomer negative (23) in a first casting dish (25), wherein the first casting dish (25) comprises a bottom (25a) with a platform (25b) that is elevated with respect to the bottom (25a), wherein the platform (25b) comprises a alignment markers (25c) for aligning the first elastomer negative (23) on the platform (25b) of the first casting dish (25), wherein the alignment markers (25c) define at least a first axis (11) for the microfluidic device (1), particularly a rotational orientation of respective extension axes of chambers (2) for receiving bioorganisms, and wherein the first casting dish (25) comprises a frame (25d) for circumferentially enclosing the bottom (25a) of the first casting dish (25), with the first set of recesses (24a) of the first elastomer negative (23) being aligned on the alignment structure (25c) of the platform (25b), wherein the frame (25d) comprises at least a section with a defined rotational orientation to said first axis (11), e. Casting a first casting compound (26) into a circumferential gap between the frame (25d) of the first casting dish (25) and the platform (25b) with the first elastomer negative (23) aligned on the platform (25b), to obtain a second casting dish (27) comprising the first elastomer negative (23) and the first casting compound (26) attached circumferentially around the first elastomer negative (23) wherein the second casting dish (27) comprises at least a portion with a defined rotational orientation to said first axis (11), f. Removing the first casting dish (25) from the second casting dish (27) to expose a main recess (27a) of the second casting dish (27) mating theu; / O 27elevation of the platform (25b) with respect to the bottom (25a) of the first casting dish (25), wherein at least one wall portion forming the main recess (27a) comprises a defined rotational orientation to said first axis (11), g. Casting a second casting compound (28) into the main recess (27a) of the second casting dish (27), to obtain a second positive master mold (29) with a second set of raised portions (29a) mating the first set of recesses (24a) of the second casting dish (27) and corresponding to the first set of raised portions (22a) of the first master mold, wherein at least one edge portion of the second positive master mold (29) comprises a defined rotational orientation to said first axis (11),h. arranging a third casting dish (30) around the second set of raised portions (29a) of the second positive master mold (29), such that the third casting dish (30) circumferentially encloses the second set of raised portions (29a) of the second positive master mold (29), wherein at least one edge portion of the third casting dish (30) comprises a defined rotational orientation to said first axis (11),i. casting the third casting dish (30) with an elastomer, particularly with PDMS (31), to obtain a first portion (100) of the microfluidic device (100) being structured on a first side according to the first elastomer negative (23) with a third set of recesses (31a) mating the first and the second set of raised portions (29a) of the first and the second positive master mold (29) wherein at least one edge portion of the elastomer, particularly of the PDMS (31), comprises a defined rotational orientation to said first axis (11), j. removing the first portion (100) of the microfluidic device (1) from the second positive master mold (29),k. attaching a second portion (200) by means of a transparent cover layer to the first side of the first portion (100) to form the microfluidic device (1).

2. The method according to claim 1, wherein the wafer (20) is a silicon wafer, a glass wafer or a plastic wafer.

3. The method according to claim 1 or 2, wherein the film (21) of curable material comprises or consists of a photoresist compound.

4. The method according to one of the preceding claims, wherein the first elastomer negative (23) comprises or consists of polydimethylsiloxane (PDMS).

5. The method according to one of the preceding claims, wherein the elastomer compound casted on the first positive master mold (22) comprises or consists of an elastomer, particularly a polyurethan elastomer, PDMS or siliconeu; / O 286. The method according to claim 4 and 5, wherein the first elastomer negative (23) and the elastomer compound casted on the first positive master mold (22) comprises or consists of an elastomer, particularly a polyurethan elastomer, PDMS or silicone.

7. The method according to one of the preceding claims, wherein the first casting dish (25) comprises or consists of plastic or metal, particularly aluminum.

8. The method according to claim 7, wherein the bottom (25a), the platform (25b) and the frame (25d) of the first casting dish (25) comprise or consist of plastic or metal, particularly aluminum.

9. The method according to one of the preceding claims, wherein the first casting compound (26) comprises or consists of an elastomer, particularly a polyurethan elastomer or silicone.

10. The method according to one of the preceding claims, wherein the second casting compound (28) comprises or consists of resin, particularly polyurethane or epoxy casting resin.

11. The method according to one of the preceding claims, wherein the third casting dish (30) comprises or consists of plastic or metal, particularly aluminum.

12. The method according to one of the preceding claims, wherein the third casting dish (30) is filled with an elastomer comprising or consisting of PDMS, such that the first portion (100) of the device (1) comprises or consists of PDMS.

13. The method according to one of the preceding claims, wherein the cover layer comprises or consists of glass.

14. The method according to one of the preceding claims, wherein the first set of raised portions (22a) defines the chambers (2) and the alignment portion (3) with the pattern (5) of markers (4) of the microfluidic device (1).

15. The method according to one of the preceding claims, wherein step b. is executed more than once, wherein each positive master mold (22) comprises a respective first set of raised portions (22a).

16. The method according to one of the preceding claims, wherein in step b., at least some of the raised portions (22a) comprise different heights.

17. The method according to one of the preceding claims, wherein step c. is executed more than once.u; / O 2918. The method according to one of the preceding claims, wherein in step d., the individual elastomer negatives (23) are aligned in a larger first casting dish (25) with a plurality of sets of alignment markers (25c).

19. The method according to claim 17 or 18, wherein each elastomer negative (23) is arranged on alignment markers (25c) associated to the respective elastomer negative (23).

20. The method according to claim 19, wherein voids between different elastomer negatives (23) are filled with additional elastomer material.

21. The method according to one of the preceding claims, wherein between steps h.and i., a lid (32) comprising at least one access hole (32a) is attached to a top side of the third casting dish (30), such that the third casting dish (30) is sandwiched between the second positive master mold (29) and the lid (32).

22. The method according to claim 21, wherein a cavity formed by the second positive master mold (29), the third casting dish (30) and the lid (32) is filled with said elastomer, particularly PDMS, via the access hole of the lid (32a).

23. The method according to claim 21 or claim 22, wherein the third casting dish (30) is sandwiched between the second positive master mold (29) and the lid (32a), particularly wherein the third casting dish (30), the second positive master mold (29) and the lid (32a) are attached to each other.

24. The method according to claim 23, wherein the attachment comprises screwing the second positive master mold (29), the third casting dish (30) and the lid (32a) together by one or more screws extending through the second positive master mold (29), the third casting dish (30) and the lid (32a).

25. The method according to claim 23, wherein the attachment comprises magnets, clamp-connections and / or elastic elements, particularly elastic bands.

26. The method according to one of the claims 23 to 25, wherein the attachment between the first portion (100) and the second portion (200) is realized by means of plasma bonding, particularly by means of an air plasma or an oxygen plasma.

27. The method according to one of the claims 22 to 26, wherein the cavity is filled completely with said elastomer.

28. The method according to one of the claims 22 to 27, wherein the elastomer filled into the cavity is cured such that the elastomer forms a permanent mechanical bond with the second positive master mold (29), the third casting dish (30) and / or the lid

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