WO2023088563A1

WO2023088563A1 - Flow cell

Info

Publication number: WO2023088563A1
Application number: PCT/EP2021/082314
Authority: WO
Inventors: Philip BÖHM; Markus WEISS-BRUMMER; Johannes Benedikt WOEHRSTEIN; Heinrich GRABMAYR
Original assignee: Deoxy Gmbh
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2023-05-25

Abstract

The present invention relates to a flow cell comprising: a core piece having a bottom side and a top side, the top side of the core piece comprising a sample section; a cover slip having a bottom side and a top side, the bottom side of the cover slip comprising a sample region; and a deformable sealing lip surrounding at least the sample section of the top side of said core piece and extending between said core piece and the bottom side of the cover slip so as to define a sealed volume between said core piece and the cover slip and so as to surround the sample region of the bottom side of the cover slip; wherein the deformable sealing lip allows for moving the sample section of the top side of said core piece relative to the cover slip between a first state with the sample section of the top side of said core piece being in contact with the sample region of the bottom side of the cover slip and a second state in which the sample section of the top side of said core piece has a first minimum distance from the sample region at the bottom side of the cover slip; wherein the sample section of the core piece comprises a micro structured section and/or wherein the sample region at the bottom side of the cover slip comprises a micro structured region.

Description

Flow Cell

The present invention relates to an actuatable flow cell, particularly for sample analysis in microscopy, wherein sample analysis may involve steps of fluid exchange and/or steps of fluid isolation. The present invention also relates to corresponding systems, microscopes and uses.

Analyzing biological samples, for example single cells, with microscopy techniques is common in biological sciences. There is a well-known technique to mount a sample on micro structured substrates for microscopic analysis. Examples may be found, e.g., in Anwar et al., Reversible sealing techniques for microdevice applications, Sensors and Actuators B 153 (2011) 301-311; in Bianco et al., Overflow microfluidic networks: application to the biochemical analysis of brain cell interactions and complex neuroinflammatory scenarios, Anal. Chem. 2012, 84, 9833- 9840; and Bose et al., Scalable microfluidics for single-cell RNA printing and sequencing; Genome Biology (2015) 16: 120), and WO2016118915. However, known devices all have drawbacks. Known devices usually require the user to perform manual assembly steps, which is inconvenient and renders these devices incompatible with automatization. Accordingly, known techniques are time consuming and require the presence of a user. Other known devices have reduced functionality, and e.g. don’t provide the possibility to fluidly isolate microcompartments, which may be beneficial, for example for performing biochemical reactions. Again other known devices do provide the possibility to fluidly isolate microcompartments, but are inherently complex, rendering them difficult to manufacture, and resulting in a low number of samples that can be analyzed in parallel.

The present invention overcomes problems that occur with known devices and techniques, at least in part.

The present invention relates to a flow cell comprising: a core piece having a bottom side and a top side, the top side of the core piece comprising a sample section; a cover slip having a bottom side and a top side, the bottom side of the cover slip comprising a sample region; and a deformable sealing lip surrounding at least the sample section of the top side of said core piece and extending between said core piece and the bottom side of the cover slip so as to define a sealed volume between said core piece and the cover slip and so as to surround the sample region of the bottom side of the cover slip; wherein the deformable sealing lip allows for moving the sample section of the top side of said core piece relative to the cover slip between a first state with the sample section of the top side of said core piece being in contact with the sample region of the bottom side of the cover slip and a second state in which the sample section of the top side of said core piece has a first minimum distance from the sample region at the bottom side of the cover slip; wherein the sample section of the core piece comprises a micro structured section and/or wherein the sample region at the bottom side of the cover slip comprises a micro structured region. Examples of samples are a cell suspension, particles in a fluid, particularly in a liquid, blood or a blood-derived sample and dissociated tissue. The flow cell may be configured such that a sample may be loaded to the sealed volume defined by the core piece, the cover slip and the sealing lip. Microscopy may preferably be performed through the cover slip. Due to its small thickness (typical thicknesses are e.g. 170 pm, or 85-115 pm), it allows for microscopy with a high magnification and/or a high numerical aperture, e.g. 60x/0.95, or 100x/1.49 magnification/numerical aperture. If the dimensions and material properties of the core piece are selected accordingly, the core piece may also be configured such that microscopy may occur via the core piece. The flow cell is preferably configured for being placed on a microscope such that an objective of the microscope may image at least a portion of the sealed volume (which may also be called sealed chamber herein), preferably via the cover slip. The flow cell is preferably configured such that an optical axis of the microscope is essentially perpendicular to the cover slip. The cover slip may allow for performing high-resolution microscopy, in which a numerical aperture of > 0.8, preferably > 0.95 is used, leading to a resolution of 0.5 pm or better over the visible spectrum.

The directional specifications herein, e.g. “bottom” and “top”, “higher” and “lower”, are to be understood as relative specifications, e.g. these specifications may be changed, as long as the respective relationships are maintained. The sample section of the top side of the core piece is that part of the core piece that defines the sealed volume. The sample region at the bottom side of the cover slip is that region of the cover slip that defines the sealed volume.

The micro structured section on the sample section of the core piece or micro structured region on the sample region at the bottom side of the cover slip or both may be configured to receive components of the sample that are to be analyzed. The micro structured region and/or section may be configured to sort, separate and/or group components of the sample. Other functions as known in the art are also contemplated.

For example, a sample may comprise fluorescently labeled cells in an aqueous solution, wherein the cells are to be analyzed with a fluorescence microscope. The sample may be loaded into the sealed chamber of the flow cell. The flow cell may be oriented such that the cells may settle into the micro structured region and/or the micro structured section due to gravity.

In the first stage, i.e. when the sample section of the top side of the core piece is in contact with the sample region of the bottom side of the cover slip, this contacting may be tight for some or all components of the sample, e.g., water-tight, fluid-tight, liquid-tight and/or particle tight. Particularly, this contacting may be tight for cells, particles, and the like that are to be analyzed. When the sample section of the top side of the core piece is in the second state, it has a first minimum distance from the sample region at the bottom side of the cover slip. In the second state the flow cell may allow for passage of some or all of the components of the sample. Whether a component of the sample can pass depends on its size as compared to the first minimum distance. If the component is smaller than the first minimum distance, it can pass, otherwise its passage is inhibited. For example, a sample may be a solution comprising cells and water. Then, the first minimum distance may be large enough to permit the passage of water but small enough to inhibit passage of the cells.

For example, the first minimum distance may be greater than 1 pm, preferably greater than 2 pm and most preferably greater than 5 pm.

The first minimum distance may be greater than 100 pm, preferably greater than 0.5 mm and most preferably greater than 1 mm. This may allow for passage of many possible sample components, e.g. many cell types, cell aggregates, etc.

Closing the flow cell, i.e. switching from the second state to the first state, may also contribute to arranging components of the sample in the micro structured section and/or region. Closing the gap between sample region and sample section may contribute to forcing components of the sample into the micro structured region and/or section, particularly when moving into the micro structured section/region requires a movement against gravity.

The flow cell is configured to change repeatedly between first and second states.

The sample section defines a plane. For example, the sample section may comprise a generally flat surface, optionally comprising the micro structured section. The generally flat surface may define a plane. In another example, the sample section may have a step-configuration, i.e. comprise a combination of generally flat surfaces at different heights, some or all of which comprise the micro structured section. In this case, the plane may be assigned to one of the flat surfaces.

The sample section may have a first stiffness in the direction orthogonal to a plane defined by the sample section greater than a second stiffness of the sealing lip in the direction orthogonal to the plane defined by the sample section. The sample section may comprise one or more materials. If the sample section comprises more than one material, the stiffness may relate to the overall stiffness. Here, the stiffness is a measure of how much resistance the piece in question offers upon applying a force in the direction specified. Assuming a linear regime, the stiffness is the linearity constant, so it can be measured by dividing the force needed to deform by the distance the piece is deformed by. In case of non-linearity, this fraction results in different values for different deformations. Therefore, here, stiffness is the fraction of deformation force and deformation distance, averaged over the deformation range to be used when applying the invention, which will be obvious to a person skilled in the art.

To test whether the first stiffness of the sample section is greater than the second stiffness of the sealing lip, the core piece can be moved between the first and the second states. The first stiffness is greater than the second stiffness for this purpose, if the sealing lip deforms more than the sample section. Thus, if an essentially flat plane of the sample section remains essentially flat when moving from the first state to the second state, the first stiffness is indeed greater than the second stiffness. The force required for moving the sample section of the top side of said core piece from its first state into its second state and/or for moving the sample section of the top side of said core piece from its second state into its first state may amount to less than 5 N, preferably less than 3 N, more preferably less than 1 N.

Here, the force may include flow cell intrinsic forces only, while e.g. the force required to overcome gravity may not be included. Alternatively, the force may include external forces, e.g. the amount for overcoming gravity. A small force is favorable for mechanical stability of the flow cell. It is also favorable in case the flow cell switches between first and second states automatically, as described below.

The deformable sealing lip may be elastic. The deformable sealing lip may have an expanded state and a compressed state. The expanded state of the deformable sealing lip may correspond to the second state. The compressed state of the deformable sealing lip may correspond to the first state. Compressed and expanded states are to be seen relative to each other. The sealing lip being elastic may mean that the sealing lip returns to the expanded state after having been in the compressed state. Particularly, it may mean that the sealing lip returns to the expanded state, when the flow cell switches from the first state to the second state, i.e. when the sample section switches from the first state to the second state.

The distance between the sample section and the sample region in the expanded state of the deformable sealing lip may amount to at least 100 pm, preferably at least 0.5 mm and more preferably to at least 1 mm. The distance may be defined as the smallest route from the sample section to the sample region.

The deformable sealing lip may be permanently, preferably adhesively attached to the core piece and/or to the cover slip. Alternatively, the deformable sealing lip may be reversibly attached to the core piece and/or to the cover slip. The deformable sealing lip may also be reversibly attached to the core piece and permanently, preferably adhesively attached to the cover slip, or vice versa.

The deformable sealing lip may seal the sealed volume by being bonded to the coverslip. The deformable sealing lip may be bonded to the cover slip in any suitable way. For example, the sealing lip may be dipped into a layer of PDMS that was spin-coated onto a substrate, thereby coating the sealing lip with a layer of PDMS. The PDMS-coating of the sealing lip is then contacted with the coverslip before hardening of the PDMS. The PDMS is allowed to harden, thus bonding sealing lip and cover slip together. This PDMS-mediated bond is sufficient to hold the core piece and its sealing lip in place relative to the cover slip. The bond may be chosen such that it is not removable during the processes described herein.

Alternatively, the deformable sealing lip may seal the sealed volume by exerting pressure onto its counterpart, e.g. the coverslip. For distance control of the core piece relative to the cover slip, and particularly of the sample section relative to the sample region, adequate mounts and/or actuators as generally known in the art may be provided. If necessary, these mounts and/or actuators may also be used to provide the pressure for sealing the sealed volume as described above.

The deformable sealing lip may be made of the same material as the core piece. The deformable sealing lip is preferably integral with the core piece. The deformable sealing lip may comprise one or a combination of the following materials: Polydimethylsiloxane (PDMS), thermoplastic elastomers (TPEs), liquid silicone rubber (LSR), polyethylene terephthalate (PET), polyamide (PA), cyclic olefin copolymer (COC).

An angle defined by the top side and, in particular, the sample section of the top side, on the one hand and the bottom side of the core piece on the other hand is smaller than 5°, preferably smaller than 3°, more preferably smaller than 2°, and even more preferably smaller than 1°. Having said angle in these defined ranges allows for a precise arrangement of the components relative to each other and relative to a microscope the flow cell is to be placed on. Particularly, it ensures that sample components that are settled into the micro structured section on the sample section of the core piece are within the reach of high numerical aperture objectives of a microscope approaching the sample from the cover slip side and that the sample section and/or sample region are in a good and defined angle with respect to the optical axis of the microscope.

The core piece is made from at least one material and the E modulus of the material of the core piece that is in contact with the coverslip/the material of the sample region and/or of the sample section may amount to 0.5-50 MPa, preferably to 1-10 MPa and more preferably to 3-6 MPa. The E modulus, which is also called elastic modulus or Young’s modulus, can be tested using standard materials science methods for tensile testing, as known in the state of the art. Having said E modulus in these defined ranges may contribute to allowing for an actuation of the core piece without too much stress to the coverslip, which would result in an overly amount of bending or even in breaking of the coverslip.

The distance between top side and bottom side of the core piece may amount to 0.25-50 mm, preferably to 0.5-25 mm and more preferably to 1-10 mm.

Having said distance in these defined ranges allows for the core piece to be flat enough to fit between optical elements, like for example condenser and objective lenses, and for dispersion of localized mechanical stress due to the elastic nature.

The area of the micro structured section and/or the area of the micro structured region may be greater than 5 mm², more preferably greater than 50 mm², even more preferably greater than 300 mm². The area may refer to the sum of all micro structured areas.

The micro structured section and/or the micro structured region may comprise microchannels and/or microwells. Any other microstructures and combinations of these microstructures are contemplated. The core piece, at least under the micro structured section, may have a total transmittance to light in the visible spectrum of at least 10 %, preferably at least 50 %, more preferably at least 90 %, and even more preferably at least 95 %.

The core piece, at least under the micro structured section, may have a phenomenological autofluorescence of less than 0.01, preferably of less than 10'³, more preferably of less than 10’ ⁴, even more preferably of less than 10'⁵. The phenomenological autofluorescence describes the relative strength of the fluorescence signal due to autofluorescence in relation to the fluorescence signal from a fluorescence standard, and depends on the fluorescent channel(s) used. For its determination, two images have to be taken: In an epifluorescence microscope (e.g. : Nikon Eclipse Ti microscope with Sola SE illumination (Lumencor, Inc), and pco.edge 4.2 camera from PCO AG, a Nikon 60x/0.95 air objective, and dichroic mirror F373-832, blue excitation/emission filters F39-480/F37-527, green excitation/emission filters F39-563/F39- 637, and red excitation/emission filters F39-640/F37-698 from AHF Analysentechnik AG. Imaging may be performed by exposing for 1 second at highest illumination intensity), acquire an image of the standard (TetraSpeck™ Fluorescent 0,1 pm Microsphere slide, ThermoFisher Scientific). The acquired image shows bright spots, which correspond to imaged microspheres. Determine the mean of the brightest pixel value of each spot (i.e. of each image of a microsphere acquired). Secondly, acquire an image with the same settings of the autofluorescence sample (e.g. the core piece), in the configuration to be used in an experiment (e.g. same position). This mage may be called autofluorescence measurement image. The phenomenological autofluorescence is defined as the quotient of the mean pixel value of an autofluorescence measurement image and the mean of the brightest pixel values of each spot in the standard image.

The core piece may comprise at least a first fluid channel extending from a first end of the fluid channel through the core piece to a second end of the fluid channel, wherein the first end of the fluid channel may located at a side of the core piece that is configured to be outside the sealed chamber, preferably at a top side or a lateral side of the core piece, and wherein the second end of the fluid channel may be located at a side of the core piece that is configured to define (i.e. be part of or adjacent to) the sealed chamber, preferably at the top side of the core piece. The first fluid channel may be configured as an inlet to the sealed chamber. A sample and or other material intended to be loaded to the sealed chamber may be loaded to the sealed chamber via the first fluid channel. The first end of the fluid channel may comprise a standardized connector, for example a Luer connector. The first fluid channel may have any suitable cross section, in terms of shape and/or in terms of size. A, preferably circular, cross section of the fluid channel may have an area of at least 0.5 mm², preferably of at least 3 mm², more preferably of at least 5 mm².

The core piece may be made from at least one core piece material and the fluid channel may be defined by a tube embedded in the at least one core piece material. Preferably, the core piece comprises a moldable material and the tube is embedded in this moldable material while the material is in a molded state. The core piece may comprise a first, so-called axis reservoir extending from the top side of the core piece into the core piece and having a dead end within the core piece. Such an axis reservoir may be configured to receive a cannula, as will be explained below.

The top end of the first axis reservoir may be adjacent to the sample section of the top side of the core piece. The first axis reservoir may be arranged to extend from the sealed chamber into the bulk of the core piece.

A, preferably circular, cross section of the first axis reservoir may have an area of at most 5 mm², preferably of at most 3 mm², more preferably of at most 0.5 mm².

The core piece may be configured to allow, preferably manual, penetration of a cannula with an outer diameter of less than 2 mm, preferably less than 1 mm, more preferably less than 0.5 mm, into the first axis reservoir. The first axis reservoir may be automatically sealed again after withdrawal of said cannula due to the elastic response of the core piece material. A particularly suitable core piece material is: Polydimethylsiloxane (PDMS), liquid silicone rubber (LSR), or thermoplastic elastomers (TPEs).

The flow cell may have at least a second fluid channel, wherein the first fluid channel is configured as an inlet of the sealed volume and wherein the second fluid channel is configured as an outlet of the sealed volume. Otherwise, the features explained with respect to the at least one first fluid channel may apply mutatis mutandis to the at least one second fluid channel.

The flow cell may have at least a second axis reservoir, wherein the first axis reservoir is configured as an inlet of the sealed volume and wherein the second axis reservoir is configured as an outlet of the sealed volume. Otherwise, the features explained with respect to the at least one first axis reservoir may apply mutatis mutandis to the at least one second axis reservoir.

The “inlet” and “outlet” may be assigned to the fluid channels and axis reservoirs in any suitable combination. For example, the first fluid channel may be configured as an inlet of the sealed volume and the first axis reservoir may be configured as an outlet of the sealed volume, or vice versa.

The flow cell may be configured such that the distance between the sample section and the sample region can be manually adjusted from 0 mm to 1 mm, preferably from 0 mm to 2 mm, more preferably from 0 mm to 3 mm.

The flow cell may further comprise a frame and the cover slip, preferably the bottom side of the cover slip, may be mounted to the frame.

The material of the frame may have an E modulus of greater than 1 GPa, preferably greater than 10 GPa, more preferably greater than 50 GPa. The frame may comprise one or a combination of the following materials: aluminum, steel, wood, polyethylene terephthalate (PET), glycol modified terephthalate (PETG), polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), acrylic styrene acrylonitrile (ASA), polypropylene (PP), polycarbonate (PC).

A thickness of the frame may amount to 0.5-10 mm, preferably to 1-8 mm, more preferably to 3-5 mm. The thickness may be measured in a direction orthogonal to the cover slip. Orthogonal to a cover slip means orthogonal to a surface of the cover slip that is generally intended for receiving a microscopy sample.

A first surface area of the cover slip, preferably of the bottom side of the cover slip, may be, preferably permanently and/or irreversibly, more preferably adhesively, bonded to a corresponding surface area of the frame. The ratio between the first surface area and the entire surface area of the bottom side of the cover slip may be at least 1%, preferably at least 10%, more preferably at least 20%.

The frame may have a, preferably circumferentially closed, opening, wherein the core piece is located within said opening. Circumferentially closed means that the opening extends, e.g. in the top-down-direction and is defined by a wall extending in top-down-direction, wherein the wall does continuously extend around the hole along a path in a plane perpendicular to the topdown-direction.

The flow cell may comprise one or more flow homogenizers between the one or more fluid channels and the micro structured section of the core piece. The one or more flow homogenizers may be arranged and shaped as generally known in the art. Preferably, the one or more flow homogenizers are arranged such that the flow of fluid at the micro structured region and/or section is sufficiently homogenous for the respective measurements.

The micro structured section of the core piece may comprise one or more elastic protrusions that extend higher, preferably at least 0.5 - 10 pm higher, than the rest of the core piece and/or the micro structured region of the cover slip may comprise one or more elastic protrusions that extend lower, preferably at least 0.5 - 10 pm lower than the rest of the cover slip. The one or more protrusions may be configured as inherent distance control.

This may allow for a defined gap between the rest of the sample region and the rest of the sample section in the first state. Stated the other way around, the elastic protrusion(s) may be used to define the gap between the rest of the sample region and the rest of the sample section in the first state. For example, the core piece may comprise several elastic protrusions that extend 10 pm higher than the rest of the core piece, while the cover slip does not comprise such protrusions. When in the first state, the elastic protrusions define the distance between the rest of the sample section and the sample region to be 10 pm or smaller, depending on the mechanical properties of the protrusions and the applied force.

The flow cell may be configured to switch between three states, namely states a, b, and c. State a corresponds to contact of the protrusions with the opposing element, i.e. in case of protrusions extending from the sample section it corresponds to contact with the coverslip and in case of protrusions extending from the sample region it corresponds to contact with the core piece. State a may also be said to be ‘open to cells’ and to ‘flushing fluids’. State b corresponds to contact of the next plane of features of the micro structured section with the coverslip and/or to contact of the next plane of features of the micro structured region with the core piece, wherein the elastic protrusions are buckled. State b may be also referred to as ‘tightly closed’. State c corresponds to the core piece and the coverslip being at a distance that there is no contact of protrusions with the opposing element. State c may correspond to a distance between the sample region and the sample section of at least 40 pm, wherein the protrusions are not taken into account for the measurement. In other words, if protrusions are present, the measurement refers to the next plane of features of the micro structured section and/or micro structured region. For example, in state the distance between the sample section and the sample region corresponds to the height of the protrusions over the next plane of features of the micro structured section or region. State c may be also referred to as ‘fully open’. Referring to any of the flow cells comprising one or more protrusions, the present invention also relates to a use of the flow cell to switch between states a, b and c.

The flow cell may comprise protrusions of different height above the sample section/below the sample region. Each height may define a defined gap between sample section and sample region as explained above with reference to protrusions of one height.

A flow cell with one or more protrusions may allow to control the distance between the sample region and the sample section with a lower need for accuracy in the angle between coverslip and core piece.

The flow cell may comprise a collecting means configured to collect fluid leaking from the sealed volume. Size and shape of the collecting means may be selected appropriately. For instance, the collecting means may be configured to be compatible with the size and shape of the rest of the flow cell as well as potentially compatible microscopes or other devices. The collecting means may be configured as a safety measure in order to avoid leaking fluid entering a microscope or any other device located in the proximity of the flow cell.

The collecting means may be a preferably transparent foil sealed to the frame. Alternatively, the collecting means may be a trough with a floor that surrounds the lower part of the flow cell, wherein the floor is preferably at least partially transparent. The trough may be configured to actuate the core piece.

The present invention also relates to a system comprising: any of the flow cells described herein, the flow cell being mounted so as to allow for performing microscopy through the cover slip of the flow cell; and a motor adapted and configured to move the core piece of the flow cell relative to the cover slip of the flow cell. The motor may be configured to adjust the distance between the sample section and the sample region in a distance range from 0 mm to 1 mm, preferably from 0 mm to 2 mm, more preferably from 0 mm to 3 mm.

The sealing lip together with the cover slip and the top side of the core piece may define a sealed enclosure for the entire distance range. “Distance range” refers to the range of the distance between the core piece and the cover slip due to possible movement of these parts relative to each other.

The system may additionally be configured for manual movement of the core piece of the flow cell relative to the cover slip of the flow cell. I.e., the distance between the sample region/cover slip and the sample section/core piece, e.g. switching between first and second states, may be performed manually by a user, e.g. via actuation of respective screws, etc.

The motor may be configured to achieve at least two, preferably three, predefined positions corresponding to at least two, preferably three, predefined distances between the sample section and the sample region. The predefined distances may correspond to contact, i.e. 0 pm, a distance in the range of 1-10 pm and a distance of greater than 40 pm. In the case of a flow cell comprising protrusions, these distances are determined by the rest of the sample region and sample section, as described above. Thus, the range l-10pm may apply for a case where elastic protrusions with a height of 1-10 pm over the next plane of features of the sample section are in contact with the coverslip. This corresponds to state a as described above. The distance of 0 pm corresponds to state b, and the distance of 40 pm corresponds to state c.

The present invention also relates to a system or any of the systems described herein, comprising any of the flow cells herein and a gimbal mount attached to the core piece. The actuation of the core piece using a gimbal mount is beneficial in that the top side of the core piece is self-aligned parallel to the coverslip on contact, needing less precise adjustment of the angle between coverslip and core piece.

The present invention also relates to a system or any of the systems described herein, comprising: any of the flow cells herein being mounted so as to allow for performing microscopy through the cover slip of the flow cell; and an (optionally ultra-) sonication device; wherein the sonication device is configured for lysing cells located in the volume between sealing lip, coverslip, and core piece by sonication.

The present invention also relates to a microscope comprising any of the systems herein.

The present invention also relates to the use of any of the flow cells herein or any of the systems herein or any of the microscopes herein for analysis of gene expression in single cells.

During the use for analysis of gene expression in single cells, a suspension of cells in a liquid may be introduced into the sealed volume of the flow cell with the sample section of the top side of said core piece being in the second state, the cells may settle into the micro structured section and/or sample region via gravity, and subsequently the sample section of the top side of said core piece may be moved to the first state, and subsequently the cells may be lysed and the gene expression may be analyzed. Subsequently the sample section of the top side of said core piece may again be moved to the second state and the sealed volume may be flushed with a liquid.

The present invention also relates to a use of any of the flow cells herein or any of the systems herein or any of the microscopes herein for live cell culturing and monitoring, wherein the core piece may optionally be gas permeable and used for CCh-supply, and wherein the sample section of the core piece may optionally be moved to the second state, preferably with the first minimum distance being in the range of 1-10 pm, to flush in buffer, and wherein the liquid may be temperature controlled.

The present invention also relates to a use of any of the flow cells herein or any of the systems herein or any of the microscopes herein for multimodal analysis of single cells, wherein different parameters of single cells may be recorded at least partially at different times, and wherein the different modalities may be connected to each other using parts of the micro structured section, or the position on the coverslip. The modalities may comprise bright field images, phase contrast images, immunofluorescent images, and/or other microscope imaging modalities. The modalities may comprise gene expression analysis as described herein.

Referring to the system comprising the sonication device, the present invention also relates to a use of the system for lysing the cells in the system by sonication.

Referring to any of the flow cells comprising one or more protrusions, the present invention also relates to a use of the flow cell to switch between contact of the protrusions with the coverslip (‘open to cells, flush fluids’), contact of the next plane of features of the micro structured region with the coverslip, wherein the elastic protrusions are buckled (‘tightly closed’), and the core piece and coverslip being at least 40 pm apart (‘fully open’).

All the features described herein with respect to the core piece/sample section/micro structured section on the one hand and the cover slip/sample region/micro structured region on the other hand may apply vice versa. Features discussed for the micro structured section may apply mutatis mutandis to the micro structured region. Features discussed for the sample section may apply mutatis mutandis to the sample region. Features discussed for the core piece may apply mutatis mutandis to the cover slip etc.

Referring to any of the systems and/or the microscope comprising the gas permeable core piece, the present invention also relates to a use of such system and/or microscope as described herein, wherein the gas permeable core piece is put into a desiccator at low pressure, e.g. at below 500 mbar, preferably below 100 mbar, more preferably below 50 mbar, before use. Such (at least partial) evacuation may help in preventing air bubbles in the micro structured region after sample application due to the following effect: Applying a liquid sample to a micro structured area may entrap air bubbles in the microstructures. However, when the core piece was previously evacuated, such entrapped air bubbles may be sucked into the material of the core piece because the core piece is at atmospheric pressure again and tends to restore the nonevacuated state and sucks in air from all sides. Any of the flow cells, systems, and microscopes herein may be used in series and/or in parallel. The present invention also relates to a system comprising two or more flow cells, wherein the system is configured for actuating the two or more flow cells individually. Alternatively or additionally, the system may be configured to actuate the flow cells simultaneously or in parallel. The flow cells may be actuated analogously, i.e. actuated such that the core pieces are in the same position relative to the cover slips.

Aspects of the invention will be discussed in the following with reference to the Figures.

Features that are discussed in the context of a specific embodiment are not restricted to this embodiment but may be combined with features that are discussed in the context of one or more other embodiments.

Figure 1 shows a schematic cross-sectional view of a flow cell;

Figures 2a, b show a schematic top view of the flow cell in Fig. 1 in views perpendicular to the view in Fig. 1;

Figures 3a-c show a schematic cross-sectional view of a flow cell in different states;

Figures 4a-c show the same flow cell and states as Fig. 3, including a sample;

Figures 5a-5d show schematic cross-sectional views of flow cells with several inlet and/or outlet features;

Figures 6a-6c show a schematic cross-sectional view of a flow cell with microstructures including protrusions in different states;

Figs 7a-7b show schematic illustrations of a combined controlling of two or more flow cells;

Figs. 8a-8b show schematic illustrations of more than one sample section zone and/or more than one sealed volume zone.

Unless stated otherwise the figures are not to scale. Particularly, relevant features, e.g. microstructures, fluid channels, axis reservoirs and sample components, may be drawn excessively large as compared to other components and distances in the drawings.

Figure 1 shows a schematic top view of a flow cell 2, which comprises a core piece 4, a frame 16, and an opening of the frame 17. Figures 2a and 2b show the flow cell 2 of figure 1 in schematic cross-sectional views along lines A-A and B-B, respectively, i.e. perpendicular to the view in Fig. 1. Figures 2a and 2b also show that the flow cell 2 comprises a cover slip 6 (cover slip 6 is not shown in Fig. 1).

The core piece 4 comprises a sample section 8 and the cover slip 6 comprises a sample region 10. The sample section 8 of the core piece is oriented towards the cover slip 6 and the sample region 10 of the cover slip is oriented towards the core piece 4. The flow cell 2 also comprises a deformable sealing lip 12. As shown in figs. 2a and 2b, the deformable sealing lip 12 may be integrally formed with the core piece 4. Alternatively, the sealing lip 12 may be, preferably permanently, more preferably adhesively, attached to the core piece 4 and/or the cover slip 6. The sealing lip 12, the sample section 8 and the sample region 10 define a sealed volume 14 due to the deformable sealing lip 12 surrounding at least the sample section 8 of the top side 4a of said core piece 4 and extending between said core piece 4 and the bottom side 6b of the cover slip 6 and so as to surround the sample region 10 of the bottom side 6b of the cover slip 6. The cover slip 6 preferably allows for microscopy through the coverslip 6, preferably for high resolution microscopy through the cover slip 6. As shown in Figs. 1 and 2a, 2b, the sample section 8 may comprise a micro structured section 8a. Although not shown, the sample region 10 may additionally or alternatively comprise a micro structured region. Although the deformable sealing lip is shown to have a specific shape, this shape is only exemplary and any suitable shape is contemplated. The same applies for the other shown components. Particularly, the micro structured section 8 may comprise any suitable structure or combination of structures selected from: one or more microwells, one or more microchannels, one or more protrusions, one or more pillars.

As indicated in the Figures with different hatching, the core piece 4 may comprise more than one material (two materials are shown, but more materials are contemplated). Particularly, the portion of the core piece 4 comprising the sample section 8 may comprise a material that is selected in view of the sample to be applied and the measurements to be made. For example, this portion may comprise a material that is transparent to light (or other types of waves and/or their wavelength). Transparent may mean that the core piece 4, at least under the micro structured section 8, may have a total transmittance to light in the visible spectrum of at least 10 %, preferably at least 50 %, more preferably at least 90 %, and even more preferably at least 95 %. The core piece, at least under the micro structured section, may have a phenomenological autofluorescence of less than 0.01, preferably of less than 10'³, more preferably of less than 10’ ⁴, even more preferably of less than 10'⁵. Both features may be taken into account in order to allow for microscopy through the material. Preferably, PDMS, TPE, LSR, COC is used. The core piece may be gas permeable. The two materials of the core piece 4 may also have different E-moduli. For example, the portion of the core piece 4 comprising the sample section 8 may comprise a material the E-modulus of which amounts to 0.5-50 MPa, preferably to 1-10 MPa and more preferably to 3-6 MPa, while the E-modulus of the other material of the core piece 4 may have a different E-modulus.

As shown in the figures, the flow cell 2 may comprise a frame 16 and the cover slip 6, preferably the bottom side 6b of the cover slip 6, may be mounted to the frame 16. As mentioned above, the material of the frame may have an E modulus of greater than 1 GPa, preferably greater than 10 GPa, more preferably greater than 50 GPa. The frame may comprise one or a combination of the following materials: aluminum, steel, wood, polyethylene terephthalate (PET), glycol modified terephthalate (PETG), polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), acrylic styrene acrylonitrile (ASA), polypropylene (PP), polycarbonate (PC). Such frame 16 may provide additional stability to the coverslip to resist forces, particularly during the first state. A thickness of the frame may amount to 0,5-10 mm, preferably to 1-8 mm, more preferably to 3-5 mm. The thickness may be measured in a direction orthogonal to the cover slip. A first surface area of the cover slip 6, preferably of the bottom side 6b of the cover slip 6b, is, preferably permanently and/or irreversibly, more preferably adhesively, bonded to a corresponding surface area of the frame 16. The ratio between the first surface area and the entire surface area of the bottom side of the cover slip may be at least 1%, preferably at least 10%, more preferably at least 20%. As indicated in the figures, the frame may have a, preferably circumferentially closed, opening 17, and the core piece 4 may be located within said opening 17.

As indicated in Figures 3a, 3b and 3c, the deformable sealing lip 12 allows for moving the sample section 8 of the top side 4a of said core piece 4 relative to the cover slip 6 between a first state (Fig. 3a) and a second state (Figs. 3b and 3c). In the first state, the sample section 8 of the top side 4a of said core piece 4 is in contact with the sample region 10 of the bottom side 6b of the cover slip 6. In the second state, the sample section 8 of the top side 4a of said core piece 4 has a first minimum distance dl from the sample region 10 at the bottom side 6b of the cover slip 6. For example, the flow cell 2 may include a first minimum distance dl greater than 1 pm, preferably greater than 2 pm and most preferably greater than 5 pm. The first minimum distance may be greater than 100 pm, preferably greater than 0.5 mm and most preferably greater than 1 mm.

The deformable sealing lip 12 may be elastic. As indicated above, this may mean that the sealing lip returns to the expanded state, when the flow cell switches from the first state to the second state, i.e. when the sample section switches from the first state to the second state.

The distance between the sample section and the sample region may be manually adjustable, e.g. from 0 mm to 1 mm, preferably from 0 mm to 2 mm, more preferably from 0 mm to 3 mm.

As indicated in Figs. 4a-c, the minimum distance dl between the micro structured section 8 and the cover slip 6 may be large enough for relevant components, e.g. fluid 28 and cells 30, of a sample in the sealed volume 14 to pass through the gap between the micro structured section 8 and the cover slip 6 (Fig. 4a, indicated by notched arrows with reference signs 28 and 30). Accordingly, the cells 30 may enter the microstructures, e.g. sink into microwells due to gravity (Fig. 4a). If the minimum distance dl is smaller than the size of the relevant components of the sample, e.g. the cells 30, these components are restricted from passing through the gap between the micro structured section 8a and the cover slip 6, as shown in fig. 4b (indicated by the crossed-out arrow). However, in this state, smaller components of the sample, e.g. fluid 28, may still pass (indicated by the notched arrow with reference sign 28). If the flow cell is switched to the first state, i.e. the micro structured section 8a is in contact with the cover slip 6 (minimum distance d 1 =0), neither cells 30 nor fluid 28, and preferably no component of the sample, may pass between sample section 8 and cover slip 6 (indicated by crossed out notched arrows).

As shown in Figs. 5a, 5c and 5d, the core piece 4 may comprise at least a first fluid channel 18 extending from a first end 18b of the fluid channel 18 at the bottom side 4b of the core piece 4 through the core piece 4 to a second end 18a of the fluid channel 18 at the top side 4a of the core piece 4. As shown in Fig. 5d, the first end 18b of the fluid channel 18 may comprise a standardized connector 19, for example a Luer connector. Any other suitable connector 19 is contemplated. Alternatively, the first end 18b may be located at another side of the core piece 4 that is configured to be outside the sealed chamber 14, e.g. at a lateral side of the core piece 4. The fluid channel 18 may have any suitable cross-section and any suitable size. For example, the shape of the cross-section may be circular and/or the cross section of the fluid channel 18 may have an area of at least 0,5 mm², preferably of at least 3 mm², more preferably of at least 5 mm².

As shown in Fig. 5b and Fig. 5c, the core piece 4 may comprise a first axis reservoir 20 extending from the top side 4a of the core piece 4 into the core piece 4 and having a dead end 20b within the core piece 4. A top end 20a of the first axis reservoir 20 may be adjacent to the sample section 8 of the top side 4a of the core piece 4.

The axis reservoir 20 may have any suitable cross-section and any suitable size. For example, the shape of the axis reservoir’s cross-section may be circular, and/or the cross section of the first axis reservoir 20 may have an area of at most 5 mm², preferably of at most 3 mm², more preferably of at most 0,5 mm².

As shown in Figs. 5b and 5d, the material of the core piece may be configured to allow penetration of a cannula 22 with an outer diameter of less than 2 mm, preferably less than 1 mm, more preferably less than 0.5 mm, into the axis reservoir 20. Preferably, the first axis reservoir 20 is automatically sealed again after and/or upon withdrawal of said cannula from the material of the core piece. This material property may be restricted to the access zone to the axis reservoir 20.

As shown in Fig. 5c, which shows a core piece 4 with a first fluid channel 18 and a first axis reservoir 20, a combination of one or more fluid channels 18 with one or more axis reservoirs 20 in one core piece is contemplated.

As shown in Fig. 5a, the flow cell 2 may have at least a second fluid channel 18. The first fluid channel 18 may be configured as an inlet to the sealed volume 14 and the second fluid channel 18 may be configured as an outlet of the sealed volume 14.

As shown in Fig. 5b, the flow cell 2 may have at least a second axis reservoir 20. The first axis reservoir 20 may be configured as an inlet to the sealed volume 14 and the second axis reservoir 20 may be configured as an outlet of the sealed volume 14.

A sample may be injected with a cannula 22 via the first axis reservoir 20 (serving as an inlet) and extracted with a cannula 22 via the second axis reservoir 20 (serving as an outlet).

A sample may be inserted via the first fluid channel 18 (serving as an inlet) and extracted via the second fluid channel 18(serving as an outlet). Any combination of fluid channel 18 and axis reservoir 20 as inlet and outlet is contemplated. For example, as shown in Fig. 5c, the first fluid channel 18 may be configured as an inlet and the first axis reservoir 20 may be configured as an outlet, or vice versa.

As shown in Fig. 5d, a cannula 22 may also penetrate into a fluid channel 18.

Figure 6a-6c each show a schematic cross-sectional view of a flow cell 2 including a micro structured section 8a including elastic protrusions 24 in the microstructure. The elastic protrusions 24 are higher than the next plane of microstructures 26 in the micro structured section 8a. Preferably, the elastic protrusions are at least 0.5 - 10 pm higher than the rest of the core piece. The shown number, positions and shapes of protrusions 24 are only exemplary and any suitable number, any suitable positions and any suitable shape are contemplated. Particularly, the properties of the elastic protrusion(s) 24 may be selected in order to tune the overall mechanical properties of the flow cell 2 and particularly the behavior during state a (contact of the protrusions 24 with the cover slip 6 but no contact of the next plane of microstructures 26 with the cover slip 6 as shown in Fig. 6a) and state b (contact of the next plane of microstructures 26 with the cover slip 6 as shown in Fig. 6b) as described above. Fig. 6c shows state c (no contact of protrusions 24 with cover slip 6, i.e. fully open). As illustrated in Figs. 6a-6c, the one or more protrusions may be configured as inherent distance control.

As illustrated in Figs 7a-7b, two or more flow cells 2 may be combined and be configured for combined controlling. Figure 7a illustrates that two or more flow cells 2 may be controlled simultaneously and/or analogously, indicated by the branched arrow 32. Figure 7b illustrates that, though being arranged in one system, two or more flow cells 2 may be controlled independently, indicated by two arrows 32.

As illustrated in Figs 8a and 8b, the sample section 8 may comprise two or more separate sample section zones 8X and 8Y. As illustrated in Fig. 8b, the sealed chamber 14 may comprise two or more chamber zones 14X and 14Y. Zones 8X, 8Y may be configured to be connected to the same sealed chamber 14 and/or chamber zone 14X, 14Y (Fig. 8a). Alternatively, the sample section zones 8X, 8Y may be configured to be connected to separate chamber zones (Fig. 8b). For more than two sample section zones 8X, 8Y, any combination of sample section zones 8X, 8Y and chamber zones 14X, 14Y may be chosen, as appropriate in view of the intended experiments.

As already mentioned above, the flow cell(s) 2 may be part of a system including a motor, the motor being configured for distance control, i.e. to control the first minimum distance.

List of reference signs:

2 flow cell;

4 core piece; 4a top side of core piece; 4b bottom side of core piece;

6 cover slip; 6b bottom side of cover slip;

8 sample section; 8a micro structured section;

8X, 8Y sample section zones; 10 sample region; 12 deformable sealing lip; 14 sealed volume/chamber;

14X, 14Y chamber zones; dl first minimum distance; 16 frame; 17 opening of frame; 18 fluid channel; a second end of fluid channel;b first end of fluid channel.; connector; axis reservoir; a top end of axis reservoir; b dead end of axis reservoir; cannula; protrusion; next plane of microstructures; fluid; cell; arrow.

Claims

Claims A flow cell comprising: a core piece having a bottom side and a top side, the top side of the core piece comprising a sample section; a cover slip having a bottom side and a top side, the bottom side of the cover slip comprising a sample region; and a deformable sealing lip surrounding at least the sample section of the top side of said core piece and extending between said core piece and the bottom side of the cover slip so as to define a sealed volume between said core piece and the cover slip and so as to surround the sample region of the bottom side of the cover slip; wherein the deformable sealing lip allows for moving the sample section of the top side of said core piece relative to the cover slip between a first state with the sample section of the top side of said core piece being in contact with the sample region of the bottom side of the cover slip and a second state in which the sample section of the top side of said core piece has a first minimum distance from the sample region at the bottom side of the cover slip; wherein the sample section of the core piece comprises a micro structured section and/or wherein the sample region at the bottom side of the cover slip comprises a micro structured region. The flow cell of claim 1, wherein the first minimum distance is greater than 1 pm, preferably greater than 2 pm and most preferably greater than 5 pm. The flow cell of claim 1, wherein the first minimum distance is greater than 100 pm, preferably greater than 0.5 mm and most preferably greater than 1 mm. The flow cell of any of the previous claims, wherein the sample section has a first stiffness in the direction orthogonal to a plane defined by the sample section greater than a second stiffness of the sealing lip in the direction orthogonal to the plane defined by the sample section. The flow cell of any of the previous claims, wherein the force required for moving the sample section of the top side of said core piece from its first state into its second state and/or for moving the sample section of the top side of said core piece from its second state into its first state amounts to less than 5 N, preferably less than 3 N, more preferably less than 1 N. The flow cell of any of the previous claims, wherein the deformable sealing lip is elastic. The flow cell of any of the previous claims, wherein the deformable sealing lip has an expanded state and a compressed state. The flow cell of claim 7, wherein the expanded state of the deformable sealing lip corresponds to the second state. The flow cell of claim 7 or 8, wherein the distance between the sample section and the sample region in the expanded state of the deformable sealing lip amounts to at least 100 pm, preferably at least 0.5 mm and more preferably to at least 1 mm. The flow cell of any of the previous claims, wherein the deformable sealing lip is permanently, preferably adhesively attached to the core piece and/or to the cover slip. The flow cell of any of the previous claims, wherein the deformable sealing lip is made of the same material as the core piece, wherein the deformable sealing lip is preferably integral with the core piece. The flow cell of any of the previous claims, wherein an angle defined by the top side and, in particular, the sample section of the top side, on the one hand and the bottom side of the core piece on the other hand is smaller than 5°, preferably smaller than 3°, more preferably smaller than 2°, and even more preferably smaller than 1°. The flow cell of any of the previous claims, wherein the core piece is made from at least one material and wherein the E modulus of the material of the core piece that is in contact with the coverslip/the material of the sample region and/or of the sample section amounts to 0.5-50 MPa, preferably to 1-10 MPa and more preferably to 3-6 MPa. The flow cell of any of the previous claims, wherein the distance between top side and bottom side of the core piece amounts to 0,25-50 mm, preferably to 0.5-25 mm and more preferably to 1-10 mm. The flow cell of any of the preceding claims, wherein the area of the micro structured section and/or the area of the micro structured region is greater than 5 mm², more preferably greater than 50 mm², even more preferably greater than 300 mm². The flow cell of any of the previous claims, wherein the micro structured section and/or the micro structured region comprise(s) microchannels and/or microwells. The flow cell of any of the previous claims, wherein the core piece, at least under the micro structured section, has a total transmittance to light in the visible spectrum of at least 10 %, preferably at least 50 %, more preferably at least 90 %, and even more preferably at least 95 %. The flow cell of any of the previous claims, wherein the core piece, at least under the micro structured section, has a phenomenological autofluorescence of less than 0.01, preferably of less than 10'³, more preferably of less than 10'⁴, even more preferably of less than 10'⁵. The flow cell of any of the previous claims, wherein the core piece comprises at least a first fluid channel extending from a first end of the fluid channel through the core piece to a second end of the fluid channel, wherein the first end of the fluid channel is located at a side of the core piece that is configured to be outside the sealed chamber, preferably at a bottom side or a lateral side of the core piece, and wherein the second end of the fluid channel is located at a side of the core piece that is configured to define the sealed chamber, preferably at the top side of the core piece. The flow cell of claim 19, wherein the first end of the fluid channel comprises a standardized connector, for example a Luer connector. The flow cell of claim 19 or 20, wherein a, preferably circular, cross section of the fluid channel has an area of at least 0,5 mm², preferably of at least 3 mm², more preferably of at least 5 mm². The flow cell of claim 19, 20 or 21, wherein the core piece is made from at least one core piece material and wherein the fluid channel is defined by a tube embedded in the at least one core piece material. The flow cell of any of the previous claims, wherein the core piece comprises a first axis reservoir extending from the top side of the core piece into the core piece and having a dead end within the core piece. The flow cell of claim 23, wherein the top end of the first axis reservoir is adjacent to the sample section of the top side of the core piece. The flow cell of claim 23 or 24, wherein the, preferably circular, cross section of the first axis reservoir has an area of at most 5 mm², preferably of at most 3 mm², more preferably of at most 0,5 mm². The flow cell of claim 23, 24, or 25, wherein the core piece is configured to allow penetration of a cannula with an outer diameter of less than 2 mm, preferably less than 1 mm, more preferably less than 0.5 mm, into the first axis reservoir and wherein the first axis reservoir is automatically sealed again after and/or upon withdrawal of said cannula. The flow cell of claim 19 to 26, having at least a second fluid channel, wherein the first fluid channel is configured as an inlet of the sealed volume and wherein the second fluid channel is configured as an outlet of the sealed volume. The flow cell of claim 23 to 27, having at least a second axis reservoir, wherein the first axis reservoir is configured as an inlet of the sealed volume and wherein the second axis reservoir is configured as an outlet of the sealed volume. The flow cell of claim 23 to 26 as depending from any one of claims 20-23, wherein the first fluid channel is configured as an inlet of the sealed volume and wherein the first axis reservoir is configured as an outlet of the sealed volume, or vice versa. The flow cell of any of the previous claims, wherein the cover slip allows for performing high-resolution microscopy. The flow cell of any of the preceding claims, wherein the distance between the sample section and the sample region can be manually adjusted from 0 mm to 1 mm, preferably from 0 mm to 2 mm, more preferably from 0 mm to 3 mm. The flow cell of any of the previous claims, wherein the flow cell further comprises a frame and wherein the cover slip, preferably the bottom side of the cover slip, is mounted to the frame. The flow cell of claim 32, wherein the material of the frame has an E modulus of greater than 1 GPa, preferably greater than 10 GPa, more preferably greater than 50 GPa. The flow cell of any of claim 32 or 33, wherein a thickness of the frame amounts to 0,5- 10 mm, preferably to 1-8 mm, more preferably to 3-5 mm. The flow cell of any of claims 32-34, wherein a first surface area of the cover slip, preferably of the bottom side of the cover slip, is, preferably permanently and/or irreversably, more preferably adhesively, bonded to a corresponding surface area of the frame. The flow cell of claim 35, wherein the ratio between the first surface area and the entire surface area of the bottom side of the cover slip is at least 1%, preferably at least 10%, more preferably at least 20%. The flow cell of any of claims 32-36, wherein the frame has a, preferably circumferentially closed, opening, and wherein the core piece is located within said opening. The flow cell of any of claims 19-37, wherein the flow cell comprises one or more flow homogenizers between the one or more fluid channels and the micro structured section of the core piece. The flow cell of any of the claims 1-38, wherein the micro structured section of the core piece comprises one or more elastic protrusions that extend higher, preferably at least 0.5 - 10 pm higher, than the rest of the core piece and/or wherein the micro structured region of the cover slip comprises one or more elastic protrusions that extend lower, preferably at least 0.5 - 10 pm lower than the rest of the cover slip. The flow cell of claim 39, wherein the one or more protrusions are configured as inherent distance control. The flow cell of any of the previous claims, comprising a collecting means configured to collect fluid leaking from the sealed volume. The flow cell of claim 41 depending from any one of claims 32-40, wherein the collecting means is a preferably transparent foil sealed to the frame. The flow cell of claim 41, wherein the collecting means is a trough with a floor that surrounds the lower part of the flow cell, wherein the floor is preferably at least partially transparent. The flow cell of claim 43, wherein the trough is configured to actuate the core piece. A system comprising: the flow cell of any of the previous claims being mounted so as to allow for performing microscopy through the cover slip of the flow cell; and a motor adapted and/or configured to move the core piece of the flow cell relative to the cover slip of the flow cell. The system of claim 45, wherein the motor is configured to adjust the distance between the sample section and the sample region in a distance range from 0 mm to 1 mm, preferably from 0 mm to 2 mm, more preferably from 0 mm to 3 mm. The system of claim 45 or 46, wherein the sealing lip together with the cover slip and the top side of the core piece defines a sealed enclosure for the entire distance range. The system of any of claims 45-47, wherein the motor is configured to achieve at least two, preferably three, predefined positions corresponding to at least two, preferably three, predefined distances between the sample section and the sample region. The system of claim 48, wherein the predefined distances correspond to contact, i.e. 0 pm, a distance in the range of 1-10 pm and a distance of greater than 40 pm. A system or any of the systems of claims 45-49, comprising: the flow cell of any of the claims 1-44; and a gimbal mount attached to the core piece A system or any of the systems of claims 45-50, comprising: the flow cell of any of claims 1-44 being mounted so as to allow for performing microscopy through the cover slip of the flow cell; and an (optionally ultra-) sonication device; wherein the sonication device is configured for lysing cells located in the volume between sealing lip, coverslip, and core piece by sonication. A microscope comprising the system of any of claims 45-51.

21 Use of the flow cell of any of claims 1-44 or the system of any of claims 45-51 or the microscope of claim 52 for analysis of gene expression in single cells. The use of claim 53, wherein, during use, a suspension of cells in a liquid is introduced into the sealed volume of the flow cell with the sample section of the top side of said core piece being in the second state, the cells are settling into the micro structured section and/or sample region via gravity, and subsequently the sample section of the top side of said core piece is moved to the first state, and subsequently the cells are lysed and the gene expression is analyzed. The use of claim 54, wherein subsequently the sample section of the top side of said core piece is again moved to the second state and the sealed volume is flushed with a liquid. Use of the flow cell of any of claims 1-44 or the system of any of the claims 45-51 or the microscope of claim 52 for live cell culturing and monitoring, wherein the core piece is optionally gas permeable and used for CCh-supply, and wherein the sample section of the core piece is optionally moved to the second state, preferably with the first minimum distance being in the range of 1-10 pm, to flush in buffer, and wherein the liquid is temperature controlled. Use of the flow cell of any of the claims 1-44 or the system of any of the claims 45-51 or the microscope of claim 52 for multimodal analysis of single cells, wherein different parameters of single cells are recorded at least partially at different times, and wherein the different modalities are connected to each other using parts of the micro structured section, or the position on the coverslip The use of claim 57, wherein the modalities comprise bright field images, phase contrast images, immunofluorescent images, and/or other microscope imaging modalities. The use of claim 57 or 58 wherein the modalities comprise gene expression analysis of any of the claims 53 to 55. Use of the system of claim 51 for lysing the cells in the system by sonication. The flow cell, system, or use of any of the preceding claims, wherein the sample section comprises two or more materials and/or the sample region comprises two or more materials. The flow cell, system, or use of any of the preceding claims, wherein the core piece is gas permeable. Use of the systems or the microscope according to claim 62, wherein the gas permeable core piece is put into a desiccator at low pressure, e.g. at below 500 mbar, preferably below 100 mbar, more preferably below 50 mbar, before use. A system or any of the systems or the microscope of the preceding claims, comprising two or more flow cells, wherein the system is configured for actuating the two or more flow cells individually. A system or any of the systems or the microscope of the preceding claims, comprising two or more flow cells, wherein the system is configured to actuate the flow cells simultaneously and/or analogously. Use of the flow cell or system or microscope as depending from any one of claims 39- 40 for switching between contact of the protrusions with the coverslip, contact of the next plane of features of the micro structured section with the coverslip, wherein the

22 elastic protrusions are buckled, and the core piece and coverslip being separated, preferably being at least 40 pm apart.

23