SYSTEM AND METHOD FOR LOADING A MICROFLUIDIC CHIP

Abstract

A method of reducing or preventing bubbles in a microfluidic sample liquid is provided. The method comprises providing a microfluidic sample holder comprising an enclosed fluid channel for holding at least part of the sample liquid, and filling at least part of the fluid channel with a sample liquid. The method further comprises: a step of pressurizing the sample liquid in the fluid channel to raise a sample liquid pressure to an elevated pressure higher than an ambient pressure and an operating pressure, a step of maintaining the sample liquid pressure at least at the elevated pressure for a predetermined period to cause dissolving of gas into the sample liquid, and a step of reducing the sample liquid pressure from the elevated pressure to the operating pressure. An associated system is also provided.

Claims

1. A method of reducing or preventing bubbles in a microfluidic sample liquid, comprising: providing a microfluidic sample holder comprising an enclosed fluid channel for holding at least part of the sample liquid, filling at least part of the fluid channel with the sample liquid, closing the fluid channel and pressurizing the sample liquid in the closed fluid channel to raise a sample liquid pressure to an elevated pressure higher than an ambient pressure and an operating pressure, maintaining the sample liquid pressure at least at the elevated pressure for a predetermined period to cause dissolving of gas into the sample liquid, and reducing the sample liquid pressure from the elevated pressure to the operating pressure.

2. The method according to claim 1, wherein the operating pressure is equal to the ambient pressure.

3. The method according to claim 1, further comprising providing an amount of gas in a limited volume in contact with the sample liquid providing a liquid level; wherein at least one of pressurizing the sample liquid in the fluid channel and maintaining the sample liquid pressure at or above the elevated pressure for a predetermined period comprises compressing the gas in the limited volume.

4. The method according to claim 1, wherein the elevated pressure is in a range of about 1-20 Bar (100-2000 kPa) over ambient pressure.

5. The method according to claim 1, further comprising adding an additional amount of sample liquid to the sample liquid.

6. The method according to claim 1, wherein the sample liquid comprises sample particles.

7. The method according to claim 1, comprising providing at least part of the fluid channel with a functionalised wall surface portion.

8. The method according to claim 1, further comprising providing a signal generator for generating an acoustic wave in the sample holder; and providing, using the signal generator, a driving signal to the sample holder generating an acoustic wave in the sample holder.

9. The method according to claim 1, further comprising providing an optical trapping beam for trapping and/or manipulating at least part of a sample in at least part of the channel.

10. A system for filling a microfluidic sample holder, wherein the sample holder comprises an enclosed microfluidic channel provided with a liquid inlet and a liquid outlet and a closure for closing the channel gas- and liquid-tight; a filling system for filling at least part of the microfluidic channel with a sample liquid; and wherein the filling system comprises a controller and a compressor and is configured for controllably pressurizing a sample liquid in the channel when closed, to raise a sample liquid pressure to an elevated pressure higher than an ambient pressure and an operating pressure, maintaining the sample liquid at the sample liquid pressure at least at the elevated pressure for a predetermined period to cause dissolving of gas into the sample liquid for removing from and/or preventing gas bubbles in the sample liquid, and controllably reducing the sample liquid pressure from the elevated pressure to the operating pressure.

11. The system according to claim 10, comprising a sealed or sealable gas reservoir for providing a defined amount of gas in a limited volume in contact with the sample liquid providing a liquid level.

12. The system according to claim 11, wherein the compressor is configured to compress a gas in the gas reservoir to pressurize the liquid.

13. The system according to claim 10, comprising a liquid reservoir in fluid connection with the channel, wherein the liquid reservoir defines a filling direction and at least part of the liquid reservoir comprises a section having an inclined wall section facing towards the filling direction.

14. The system according to claim 10, wherein the sample holder comprises a translucent or transparent portion for optical detection of a liquid level, and wherein the translucent or transparent portion may be provided with a level mark and/or allow optical access to a level mark, and/or at least part of the translucent or transparent portion is formed as a light guide.

15. The system according to claim 10, wherein the sample holder comprises an at least partly opaque portion at or near the liquid inlet and/or the liquid outlet, wherein the opaque portion provides a translucent or transparent portion and/or an aperture, for lighting at least one of a liquid inlet of the channel and a liquid outlet of the channel; wherein in particular in a system according to claim 12 the translucent or transparent portion and/or an aperture is separate from the translucent or transparent portion for optical detection.

16. The system according to claim 13, comprising a sealed or sealable gas reservoir for providing a defined amount of gas in a limited volume in contact with the sample liquid providing a liquid level; and a closure for closing the gas reservoir and/or the liquid reservoir.

17. The system according to claim 10, wherein the sample holder comprises an acoustic wave generator for generating an acoustic wave in at least part of the channel, and/or the system comprises at least one optical tweezer for trapping and/or manipulating at least part of a sample in at least part of the channel.

18. The system according to claim 10, wherein the elevated pressure is in a range of about 1-20 Bar (100-2000 kPa) over ambient press.

19. The method according to claim 8, wherein generating the acoustic wave in the sample holder provides an acoustic force in at least part of the channel for manipulating one or more objects in the sample liquid.

20. The system according to claim 13, wherein the inclined wall section defines a first section having a first inclination and a second section having a second, different inclination.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The above-described aspects will hereafter be more explained with further details and benefits with reference to the drawings showing a number of embodiments by way of example.

[0075] FIG. 1 is a schematic drawing of an embodiment of a manipulation system;

[0076] FIG. 2 is a schematic drawing of a sample holder for the system of FIG. 1;

[0077] FIG. 2A is a schematic detail of FIG. 2 as indicated;

[0078] FIGS. 3-11 show an exemplary workflow according to the presently provided concepts;

[0079] FIG. 12 is a schematic overview of method steps according to an embodiment;

[0080] FIG. 13 shows an exemplary sample holder assembly for the system and method in perspective;

[0081] FIG. 14 is a side view of the sample holder 300;

[0082] FIG. 15 is a bottom view of the sample holder 300;

[0083] FIG. 16 is a perspective cross section view of the sample holder assembly of FIGS. 13-15;

[0084] FIG. 17 shows part of the sample holder assembly of FIGS. 13-15;

[0085] FIG. 18 is a view like FIG. 17, but partly cut away;

[0086] FIG. 19 indicates a schematic cross section profile of the reservoir.

DETAILED DESCRIPTION OF EMBODIMENTS

[0087] It is noted that the drawings are schematic, not necessarily to scale and that details that are not required for understanding the present invention may have been omitted. The terms “upward”, “downward”, “below”, “above”, and the like relate to the embodiments as oriented in the drawings, unless otherwise specified. Further, elements that are at least substantially identical or that perform an at least substantially identical function are denoted by the same numeral, where helpful individualised with alphabetic suffixes.

[0088] Further, unless otherwise specified, terms like “detachable” and “removably connected” are intended to mean that respective parts may be disconnected essentially without damage or destruction of either part, e.g. excluding structures in which the parts are integral (e.g. welded or moulded as one piece), but including structures in which parts are attached by or as mated connectors, fasteners, releasable self-fastening features, etc. The verb “to facilitate” is intended to mean “to make easier and/or less complicated”, rather than “to enable”.

[0089] FIG. 1 is a schematic drawing of a manipulation system 1, FIG. 2 is a cross section of a sample holder and FIG. 2A is a detail of the sample holder of FIG. 2 as indicated with “IIA”.

[0090] The system 1 comprises a microfluidic sample holder 3 comprising a fluid channel 4 providing a holding space 5 for holding a microfluidic sample 7. As shown in FIG. 2A and further, the sample may typically comprise one or more objects like biological cellular bodies 9, 10 in a sample liquid 11 as exemplary objects of interest. It is noted that also, or alternatively, other types of objects like microspheres could be used, possibly attached to biological cellular bodies 9. The system 1 further comprises an acoustic wave generator 13, e.g. a piezo element, connected with the sample holder 3 to generate an acoustic wave in the holding space 5 exerting a force on the sample 7 and cellular bodies 9, 10 in the sample 7. The acoustic wave generator 13 is connected with an optional controller 14 and power supply, here as an option being integrated.

[0091] The sample holder 3 comprises a wall 15 providing the holding space 5 with an optional functionalized wall surface portion 17 to be contacted, in use, by part of the sample 7. Here, the functionalized wall surface portion 17 is provided with the cellular bodies 10 adhered to the surface of the wall 15, possibly with one or more primer layers in between (not shown). As explained in detail below, interaction of the cellular bodies 9 (and/or other objects) with the cellular bodies 10 may be studied with such system. A further wall, e.g. opposite wall 16, may also or alternatively be provided with a (further) functionalized wall surface portion.

[0092] The shown manipulation system 1 comprises a microscope 19 with an optional optical system such as an (optionally adjustable) objective 21 and a camera 23 connected with a computer 25 comprising a controller and a memory 26; more or less optical detectors and/or detectors of other types may be provided. The computer 25 may also be programmed for tracking one or more of the cellular bodies based on signals from the camera 23 and/or for performing microscopy calculations and/or for performing analysis associated with (super resolution) microscopy and/or video tracking, which may be sub-pixel video tracking. The computer or another controller (not shown) may be connected with other parts of the system 1 (not shown) for controlling at least part of the microscope 19 and/or another detector (not shown). In particular, the computer 25 may be connected with one or more of the acoustic wave generator 13, the power supply thereof and the controller 14 thereof, as shown in FIG. 1. The computer may also be connected to one or more fluidics valves, pressure- and/or flow sensors, pressure- and/or flow regulators, etc. in order to facilitate control over sample liquid flows.

[0093] The system further comprises an optional light source 27. The light source 27 may illuminate the sample 7 using any suitable optics (not shown) to provide a desired illumination intensity and intensity pattern, e.g. plane wave illumination, Köhler illumination, etc., known per se. Here, in the system light 31 emitted from the light source 27 is directed through the acoustic wave generator 13 to (the sample 7 in) the sample holder 3 and sample light 33 from the sample 7 is transmitted through the objective 21 and through an optional ocular 22 and/or other optional optics (not shown) to the camera 23.

[0094] The sample light 33 may comprise light 31 affected by the sample (e.g. scattered and/or absorbed) and/or light emitted by one or more portions of the sample 7 itself e.g. by fluorophores attached to the cellular bodies 9 or e.g. generated by bio-, or chemo-luminescence.

[0095] As shown in FIGS. 1 and 2, the sample holder 3 may comprise a part 3A that has a recess being, at least locally, generally U-shaped in cross section and a cover part 3B to cover and close (the recess in) the U shaped part providing a channel 4 and a holding space 5 enclosed in cross section.

[0096] The sample holder 3 is a microfluidic device of the type commonly referred to as a lab-on-a-chip. The sample holder may be a substantially planar device. At least part of the sample holder may be formed by a single piece of material with the channel 4 inside, e.g. glass, injection moulded polymer, etc. (not shown) or by fixing different layers of suitable materials together more or less permanently, e.g. by welding, glass bonding/direct bonding, gluing, taping, clamping, etc., such that the channel 4 and holding space 5 are formed in which the sample 7 may be contained, at least during the duration of an experiment. Forming the sample holder from a single piece of material may have the advantage that it forms an efficient acoustic cavity which enables the generation of high acoustic forces at the functionalized wall. Thus, a monolithic sample holder, at least at the location of the acoustic wave generator 13, may be preferred over an assembled sample holder for improving acoustic coupling, reducing losses and/or preventing local variations.

[0097] Embodiments of a sample holder 3 will be detailed below.

[0098] As shown in FIG. 2, the sample holder 3 is connectable to a fluid flow system 35 for introducing fluid into the holding space 5 of the sample holder 3 and/or removing liquid from the holding space 5, e.g. for flowing liquid through the channel 4 and holding space 5 (see arrows in FIG. 2).

[0099] The fluid flow system 35 may comprise a manipulation and/or control system, possibly associated with the computer 25. The fluid flow system 35 may comprise one or more of reservoirs 37, pumps, valves, and inlet conduits 38 for introducing one or more liquids and outlet conduits 39 for removing one or more liquids, sequentially and/or simultaneously. The sample holder 3 and the fluid flow system 35 may comprise connectors, which may be arranged on any suitable location on the sample holder 3, for coupling/decoupling without damaging at least one of the parts 3, 35, and preferably for repeated coupling/decoupling such that one or both parts 3, 35 may be reusable thereafter. Further, an optional machine-readable mark M or other identifier is attached to the sample holder 3, possibly comprising a memory.

[0100] FIG. 2A is a schematic of cellular bodies 9 in the sample holder 3 of FIG. 2. Part of the wall 15 of the sample holder 3 is optionally provided with a functionalized wall portion 17, e.g. an area of the area being covered with biological cells 10 of a different type to which the cellular bodies of interest 9 may adhere. Also shown is part of the microscope lens 21 and an optional immersion fluid layer FL for improving image quality.

[0101] On providing a periodic driving signal to the acoustic wave generator 13 a standing wave is generated in the sample holder 3. The standing wave exerts an acoustic force on objects 9, 10 in the sample liquid 11 having a different compressibility (also referred to as acoustic index) than the surrounding sample liquid 11. The signal is selected such that an antinode of the wave is generated at or close to the wall surface (of the sample holder 3 e.g. surface portion 17) and a node N of the wave away from the surface 17, generating a local maximum force on the bodies 9, 10 at or near the surface towards the node. Thus, as explained in detail in WO 2018/083193, incorporated herein by reference, application of the signal may serve to probe adhesion/detachment of the bodies 9 to the surface and/or objects 10 etc. on the surface in dependence of the strength of the force.

[0102] FIGS. 3-5 indicate a top view of a sample holder 3 and indicate exemplary method steps. As indicated in FIGS. 3-5, an acoustic force sample holder 3 may comprise a single channel 4. The holding space 5 may be determined by a shape variation in the channel 4, and/or by the location of the acoustic wave generator 13 and/or by the location of a window for imaging etc. overlapping part of the channel 4. In the shown embodiment, the channel 4 comprises an inlet 41 and an outlet 43 for connection to the conduits 38, 39 (cf. FIGS. 1-2).

[0103] FIG. 3A indicates filling the channel 4 and the holding space 5 with a sample liquid via the inlet 41, e.g. using the conduit 38 and/or another liquid supply 47 such as a pipette. Thus, at least part of the channel 4 is filled with the sample liquid 11. The filling may be done by allowing the sample liquid to flow into the channel 4 by gravity and/or by wetting- and/or capillary action of the channel 4 alone. Also or alternatively, at least part of the filling may be done by pressure (difference) such as by applying pressure at the inlet 41 and/or suction at the outlet 43. Preferably, the sample fills the sample channel 4 completely. When filling, bubbles may be prevented as much as possible by adjusting a flow velocity and/or by flushing accidental bubbles out of the channel 4.

[0104] During and/or after the loading, the sample liquid pressure may be ambient pressure P1.

[0105] FIG. 3B shows that, in a subsequent step, the liquid in the channel 4 is pressurised for a first time to a first elevated pressure P2, higher than the ambient pressure P1, using a compressor 50. At the outlet 43 a closure is provided to allow the pressure build-up in the channel 4 and prevent pushing sample liquid out of the channel 4. The compressor 50 may comprise a piston 51, and in the compressor and/or in an optional conduit between the compressor and the inlet 41 (not shown) an amount of gas may be provided determining a liquid level LL. The compressor may then operate on the gas to compress the gas and thereby cause increasing the sample liquid pressure to the elevated pressure P2. The sample liquid pressure is maintained at the elevated pressure P2 for a period of time, e.g. 3-5 hours, to cause dissolving of gas into the sample liquid and to destroy any bubbles. The elevated pressure P2 need not be (held) constant. Removal of the bubbles may be determined by monitoring presence and/or size of bubbles in the holding space 5. When bubbles are not or no longer detected, the sample pressure may be reduced to an operating pressure.

[0106] Establishing and maintaining the sample liquid pressure at an elevated pressure may be repeated; it is conceivable that after an initial period of maintaining the elevated pressure, the pressure is reduced but that bubbles considered to have been destroyed grow and/or reappear again to become detectable at a pressure at or near the operating pressure. Repetition of the steps of pressurising the sample liquid and maintaining the sample liquid pressure at an elevated pressure may ensure removal of all bubbles or at least all bubbles negatively affecting the sample and/or use of the sample.

[0107] FIG. 4 and FIG. 5 indicate subsequent loading the channel 4 and the holding space 5 with a further sample liquid and optional target cells 10 via the inlet 41, e.g. using the conduit 38 and/or another liquid supply 47 such as a pipette. Thus, at least part of the channel 4 is filled with the sample liquid 11. The further sample liquid in this step may be different from the sample liquid used in the step before, but preferably being substantially the same and/or being at least miscible with the sample liquid already in the channel 4. The filling may again be done by allowing the sample liquid to flow into the channel 4 by gravity. Also or alternatively, at least part of the filling may be done by pressure (difference) such as by applying pressure at the inlet 41 and/or suction at the outlet 43. When filling, bubbles may be prevented as much as possible by adjusting a flow velocity and/or by flushing accidental bubbles out of the channel 4.

[0108] During and after the subsequent loading, the target cells 10 entrained in the sample liquid filling and flowing through the sample channel 4 are distributed over the channel 4 and the holding space 5 and left to settle there on a wall of the sample holder 3. Thus, a functionalized wall surface portion 17 is formed, see FIGS. 5 and 6 cf. FIG. 2A. Formation of the functionalized wall surface portion 17 may comprise further suitable steps such as giving the target cells 10 time to attach to the surface and grow into a monolayer. Also, a functionalized wall surface portion 17 may (be formed to) comprise different portions and/or portions with different properties.

[0109] The functionalized wall surface portion 17 may be distributed over the channel 4 beyond the holding space 5, and may extend into conduits 38 and/or 39 if used. The loaded cells 10 may be incubated and/or cultured in the channel 4. At least part of the medium may be refreshed during incubation/culturing. Flow rates over a few tens of nanoliters per minute in microfluidic channels and/or shear stress levels over 1 mPa may be avoided to reduce or prevent shear stress to the cells.

[0110] During and/or after the loading, the sample liquid pressure may be ambient pressure P1.

[0111] FIG. 6 shows that, after the loading and possibly during the incubation and/or culturing, the liquid in the channel 4 may optionally be pressurised a second time to a second elevated pressure P2-1, higher than the ambient pressure P1, using a compressor 50 (possibly the same as before), and that the sample pressure is maintained at the second elevated pressure P2-2 for a second period of time, e.g. half an hour or 1 hour. Thereafter the sample liquid pressure is reduced to a second operating pressure P3-2 which may be equal to an ambient pressure and/or the (first) operating pressure P3. The second elevated pressure P2-2 may be equal to or different from the first elevated pressure P2.

[0112] FIG. 7 indicates, as a subsequent step, loading a further sample component comprising objects 9 such as effector cells 9 or other cellular bodies in a sample liquid into the holding space 5 from a supply 49 and/or conduit 38 via the channel 4 (see arrows). During the loading, care may be taken to fill not only a lead-in section 4A of the channel 4 upstream of the holding space 5 but also a lead-out section 4B of the channel 4 downstream of the holding space 5 to ensure that the cells 9 pass through and into the holding space 5. Note that in prior art experiments, bubbles would often have been present in the channel 4 and such bubbles were found to become entrained in the fluid flow and cause damage to the functionalized layer of target cells 10. Such problems are prevented with the present concepts.

[0113] FIG. 8 indicates that the effector cells 9 may be allowed to settle and/or interact with the target cells 10. Again, the thus prepared sample may be stored and/or cultured in the channel 4 with or without (sample) liquid flow.

[0114] FIG. 9 indicates that, as explained before with reference to FIG. 6, the sample fluid may optionally be pressurised a third time to raise a sample liquid pressure to a third elevated pressure P2-3, and that the sample pressure is maintained at the third elevated pressure P2-3 for a third period of time, e.g. half an hour or 1 hour. Thereafter the sample liquid pressure is reduced to a third operating pressure P3-3 which may be equal to an ambient pressure and/or the (first or second) operating pressure. The third elevated pressure P2-3 may be equal to or different from the first and/or second elevated pressures P2, P2-2.

[0115] Different embodiments may comprise only a single instance of such combination of steps of pressuring the sample liquid to an elevated pressure and maintaining the elevated pressure, or two instances or rather even more such instances, in particular in case sample liquid is added and/or exchanged more times. However, multiple instances, i.e. re-application of pressure and maintaining such pressure, may not be necessary if addition of new fluids is done in a properly wetted fluid reservoir and/or if trapping of gas is avoided, this may be facilitated if sharp corners and/or rough surfaces are prevented in the reservoir where the new fluid may be introduced. Some sharp corners and rough surfaces may be difficult to avoid in the full microfluidics system, in particular for example at the interfaces between the chip and the rest of the fluidics system. An advantage of the current method is that as long as the system is fully wetted once by pressurization it may be possible to avoid or limit bubble formation at later steps without the need for further pressurization and/or other measures. This leads to more design freedom for microfluidics systems.

[0116] FIGS. 10-11 indicate exemplary use of the sample holder 3 and sample at an operating pressure P3. In particular, FIG. 10 indicates generating an acoustic wave in the holding space 5 by the acoustic wave generator 13 connected with the sample holder 3, thus exerting a force on the cellular bodies 9, 10 of the sample in the holding space 5. The acoustic force is, as a preferred option, adjusted such that part of the cells 9 are forced from the functionalized wall surface 17 towards the node in the sample liquid (indicated as cells 9′ with a lighter color), while cells 9 that are stronger bound to the functionalized wall surface 17 may remain adhered (cf. FIG. 2A). Thus, by adjustment of the force, separations may be made between different types of effector cells 9 based on adhesion characteristics; e.g. unbound—loosely bound—strongly bound.

[0117] FIG. 11, e.g., shows that the detached cells 9 may be flushed out of the holding space 5 and removed from the sample holder 3 at the outlet 43 and collected. If so desired, thereafter a further acoustic wave may be provided in the holding space 5, e.g. stronger than the first to detach stronger bound cells not detached before, and such later-detached cells 9 (i.e. having been more strongly bound) may be separately collected.

[0118] Note that the terms “inlet” and “outlet” may generally relate to the direction of a fluid flow through the respective structure, rather than specific ports of the sample holder 3, unless one or more one-way flow direction elements (valves, pumps, etc. are provided). E.g., in a variant to the process described above with respect to FIGS. 10-11, during or after application of the acoustic wave, a fluid flow direction may be reversed and the inlet 41 may serve as outlet, whereas outlet 43 may serve as inlet for liquid.

[0119] FIG. 12 is a schematic overview of method steps according to an embodiment, identified as steps S1-S6. [0120] Step S1 indicates a step of providing a microfluidic sample holder comprising an enclosed fluid channel for holding at least part of the sample liquid. [0121] Step S2 indicates filling at least part of the fluid channel with a sample liquid. The sample liquid thereafter is provided at a first pressure P1, e.g. ambient pressure. [0122] Step S3 indicates pressurizing the sample liquid in the fluid channel to raise a sample liquid pressure to an elevated pressure P2 higher than the first pressure P1 higher than an ambient pressure and an operating pressure. [0123] Step S4 indicates maintaining the sample liquid pressure at least at the elevated pressure for a predetermined period to cause dissolving of gas into the sample liquid. [0124] Step S5 indicates reducing the sample liquid pressure from the elevated pressure to the operating pressure. [0125] Step S6 indicates use of the sample comprising the sample liquid, having had any bubbles in it removed, at an operating pressure P3. An exemplary use comprises manipulating one or more objects in the sample liquid by an acoustic force as described herein elsewhere.

[0126] As discussed above with respect to FIGS. 3A-3B, 6 and 9, steps S2-S5 may be repeated one or more times, wherein step S2 may then comprise modifying the sample such as by changing an amount and/or composition of the sample, e.g. adding and/or exchanging sample liquid with respect to previous instances of performing step S2, and/or introducing sample particles, cf. FIGS. 4-5 and/or FIG. 7.

[0127] FIG. 13 shows an exemplary sample holder 300 for the system and method in perspective.

[0128] FIG. 14 is a side view of the sample holder 300.

[0129] FIG. 15 is a bottom view of the sample holder 300.

[0130] The sample holder 300 comprises a “chip” 303 in a housing 350. FIG. 15A is a detail of FIG. 15.

[0131] The shown housing 350 comprises a bottom shell 351 and an upper shell 353, which here comprises two parts, referred to as chip cover 355, and connector part 357, respectively. The housing 350 holds the chip 303.

[0132] The parts 351, 353 (=355, 357) are attached together around the chip 303, e.g. using bolts 358 as indicated, but other attachment systems could be used, e.g. clamps, and/or be permanently attached, e.g. glued or welded. It is noted that a suitable housing could comprise more or less parts and each part and/or the housing as a whole could be shaped differently than shown here. The housing 350 may be at least partly opaque. Screw bolts 359 are provided as one option for fixing the sample holder 300 to other parts of the system (not shown).

[0133] FIG. 16 is a perspective cross section view of the sample holder assembly of FIGS. 13-15, showing the sample holder or “chip” 303 in the housing 350. Also visible is that the housing 350 further accommodates an optional printed circuit board 561 (“PCB”) and an optional connector pad 363. The PCB 361 may provide any electrical connection to the chip and/or may provide other functions such as chip ID, calibration parameters and temperature control. The connector pad 363 may facilitate a thermal connection between the chip and the PCB 351. A multi-pin electrical connector 365 is provided for connecting control- and/or power signals to an optional acoustic transducer on the chip 303, (see also FIG. 15) and/or for other signals for one or more of control, power, temperature control, detection and measurement.

[0134] FIGS. 13, 15 and 16 show that the chip cover 355 comprises a first window 373 and that the bottom shell 351 of the housing 350 comprises two windows 375 and 377, respectively. The windows 373, 375 are arranged overlapping, possibly registered, and allow approaching of and/or contact and/or optical access to the chip 303 (see also below) as well as allowing that the housing 350 is opaque elsewhere. However, in the shown embodiment, the connector part 357 is transparent. The windows 373, 375, 377 are optional and if present may be formed as openings, as here, and/or one or more of them may comprise a transparent portion.

[0135] FIG. 15A is a detail of FIG. 15, as indicated, and shows that the window 377 provides optical access to and/or illumination of part of the chip 303, in particular the inlet 341 and outlet 343 of a channel 304 in the chip 303, see below.

[0136] FIG. 17 shows the connector part 357 of the housing 350 and the chip 303, without the bottom shell 351 and chip cover 355. Between the connector part 357 and the chip 303 a resilient sealing gasket 379 is arranged, providing a liquid and gas tight connection between the connector part 357 and the chip 303. In dashed lines, some (not all) structures are indicated which are internal in the connector part 355, the gasket 379 and the sample holder 303, respectively.

[0137] FIG. 18 is a view like FIG. 17, but now with part of the connector part 357 cut away.

[0138] In the chip 303 a fluid channel 304 is indicated. The chip 303 may be, as shown, generally planar and the channel 304 is generally U-shaped in such plane. The channel 304 comprises a widened portion 305 which forms a holding space for a sample for experiments. The (channel 304 of) chip 303 comprises an inlet 341 and an outlet 343 for fluid sample materials. The sample holder 303 further is provided with an acoustic wave generator 313 such as a piezo element or other transducer for generating an acoustic wave in the holding space 305. FIGS. 17 and 18 also show electrical connections 380 for the acoustic wave generator 313.

[0139] The connector part 357 comprises a sample liquid reservoir 381 fluidly connected with the inlet 341 of (the channel 304 of) the chip 303. The liquid reservoir 381 is closeable gas tight with a sealed cap closure 382 (see also FIGS. 13, 14). Further, the connector part 357 provides a conduit 339 fluidly connected with the outlet 343 of (the channel 304 of) the chip 303, here via an optional ferrule 339A contained in or by the gasket 379, providing a liquid and gas tight seal (see also FIG. 16A).

[0140] Referring again to FIGS. 13-16, attached to the housing 350 is an optional mount 383 holding a valve 384. The valve 384 is connected with the chip 303 via the conduit 339.

[0141] A syringe 385, or other fluid reservoir, may be connected with the valve 384 as shown, preferably releasably connected. The syringe 385 comprises a cylinder 386 and a piston 387. In the shown embodiment, the syringe 385 is provided with an optional adjustable clamp 391. The clamp 391 and the syringe 385 are attached to each other, preferably removably attached. The shown exemplary clamp 391 comprises a mount 393 and a pusher 395 threaded into the mount 393. When the clamp 391 and the syringe 385 are operably assembled as shown, the clamp 391 can controllably depress the piston 387 into the cylinder 386 of the syringe 385 by screwing the pusher 395 into or out of the mount 393. Likewise, also or alternatively a desired relative position of the piston 387 and the cylinder 386 may be established and maintained. The assembly of the syringe 385 and the clamp 391 serves as an adjustable compressor as will be set out below.

[0142] Referring back again to FIGS. 17-18 and now also referring to FIG. 19, indicating a schematic cross section profile of the reservoir 381, the reservoir 381 defines a filling direction FD, in particular wherein the reservoir 381 is substantially vertically pointing upward and widening for receiving a pipette tip (not shown) or the like for filling the reservoir with a sample liquid when the cap 382 is open or removed (cf. FIG. 17, 18). Thus, the liquid reservoir 381 comprises a tapering section having an inclined wall section facing towards the filling direction. In particular, the tapering section defines a first section 397 having a first tapering angle and a second section 399 having a second, different, tapering angle, respectively.

[0143] The first section 397 allows for easy filling as indicated above and possibly for holding relatively large amounts of liquid. This may also facilitate rinsing of the reservoir 381 (and possibly of the channel 304), e.g. for consecutive addition of different liquids, for working with valuable sample materials and/or for cleaning and reuse.

[0144] The second, relatively narrow and steep section 399 facilitates release of bubbles by the tapering shape. In the comparably narrow portion 399 small volume changes in the reservoir 381 are easier noticeable than in a comparably wider portion 397. This facilitates determining small volumes and/or filling the sample holder 303 with such small volumes (typically on the order of about 10 microliters) such as for working with valuable sample materials, e.g. (a sample comprising) patient extracted T-cells. Also, because of the steeper taper of this second section 399, for a given fluid flux there is a relatively small change in the speed of movement of the liquid level (e.g. meniscus) as the liquid level drops or rises in the second section 399, compared to in the first section 397 that is wider and has less steep walls. E.g. during emptying of the reservoir, due to the steep taper of the second section 399 there is only a small acceleration of the liquid level drop as the liquid level approaches the minimum visible liquid level height. This facilitates better control over the liquid level by the user. Bubbles introduced by pipetting or nucleation may be removed by releasing from the wall. Bubbles may release better due to the inclined wall upward facing the liquid level in the filling direction provided by the slight taper, compared to a vertical wall such as in a non-tapered straight reservoir. This may be because chances are reduced that a bubble contacts the wall just released and/or, as the bubble in contact with the reservoir wall rises up there is a smaller chance to contact the opposite wall. This facilitates bubble release. The taper, of the first and/or of the second section may also facilitate guiding a filling device such as a pipette tip to the inlet 341 of the sample holder 303 for delivery of sample material close to the sample holder. This may reduce accumulation or remaining of sample objects in the reservoir 381 which might interfere with measurements and/or manipulation in the channel 304 later on, e.g. when further sample liquids are introduced.

[0145] The reservoir 381 may be provided with one or more level marks M for reference (FIG. 18).

[0146] The connector part 357 provides a window 401 for optical detection, in particular visual detection, of a liquid level and/or a level mark in the reservoir 381. The window 401 also allows the user or the system to detect potential bubble issues, in particular by allowing inspection close to the bottom of the reservoir and/or the inlet hole 341 of the chip. For that, at least part of the connector part 357 is transparent, possibly all of the connector part 357, as in the shown embodiment. Preferably most of the reservoir 381 if not all of it is visible through the window 401. The window 401 may be plane or be curved or otherwise formed to provide lens action for magnification and/or otherwise facilitating detecting a liquid level in the reservoir. The orientation of the window 401 and/or further more or less conspicuous optical indicators may urge a user to adopt a predetermined viewing angle and/or direction, thus increasing consistency between detections and reliability of the procedure.

[0147] Due to the translucency and/or transparency of the connector part 357 level indication is facilitated, which may be further assisted by the window 377 enabling access of light “from below”.

[0148] An exemplary method of filling the channel 304 of the sample chip 303 in the sample holder 300 comprises the following steps: [0149] A—Filling the syringe 385 with a known volume of a gas, e.g. 0.5 ml of air. The syringe 385 may be detached from the valve 384 for this. The syringe 385 should then be connected with the valve. Note that a three-way valve (or more than three ways) could be used for filling the syringe with another gas and/or for filling the syringe 385 without removal from the valve 384. [0150] B—Filling the liquid reservoir 381 with a known quantity of a sample liquid. E.g., 0.3 ml of aqueous solution Phosphate Buffered Saline (PBS). The known quantity may be determined by a level mark in the reservoir 381 and/or by using a calibrated pipette. Preferably, bubble formation during filling is prevented or minimised. [0151] C—Filling the sample channel 304 with the sample liquid. In the present method this is done by suction of the liquid through the channel using the syringe 385 by retracting the piston (and setting the valve 384 open between the syringe 385 and the sample holder 303). The desired amount of retraction may be determined from indications on the syringe but it is preferred that (receding of) a liquid level in the reservoir 381 to a predetermined height (possibly indicated with a level mark on or in (the reservoir 381 of) the system 300 is used as a gauge. The resultant syringe configuration is maintained thereafter until in step F below. [0152] D—Re-filling the liquid reservoir 381 with a further known quantity of a sample liquid. E.g., again 0.3 ml of aqueous solution PBS. The known quantity may be determined by a level mark in the reservoir 381. Preferably, bubble formation is prevented or minimised. The valve 384 may remain open or be closed between the syringe 385 and the chip 303. [0153] E—Close the reservoir 381 liquid- and gas tight with the sealing cap 382. The valve 384 may be open or closed. [0154] F—Pressurize the sample liquid in the fluid channel 304 using the syringe 385 provided with the clamp 391. This may comprise one or more of the following sub-steps: [0155] F1—Ensure that the conduit is liquid-filled and that a liquid level in the syringe 385 perpendicular to the conduit entrance, e.g. by orienting the syringe vertical with the piston up and the syringe-outlet down, so that no bubbles are accidentally introduced into a conduit and/or the channel. [0156] F2—If not already done so previously, attach the clamp 391 to the syringe 385. [0157] F3—While maintaining the orientation discussed in substep F1, with the valve 384 open compress the gas in the syringe 385 by depressing the piston 387 for a predetermined amount and fixing the piston 387 at the desired position to pressurise and maintain the gas at the thus established desired elevated pressure. Note that by closing off of the reservoir 381 in substep E the pressure may be built up since the opposite ends of the sample holder are sealed. In case of a sample holder comprising plural interconnected channels inside the chip, all in- and outlets should be sealed liquid- and gas tight for the pressurization.

[0158] Note that pressurisation may be applied from either an inlet-side or an outlet-side of the channel. E.g. pressurisation could also be provided from the side of or on the reservoir. In such case, the same or a further assembly comprising a syringe and a clamp could be connected with (a modified version of) the cap 382 (not shown), possibly via a suitable port on a multi-port valve replacing the shown valve 384. [0159] G—Maintaining the sample liquid pressure at least at the elevated pressure for a predetermined period to cause dissolving of gas into the sample liquid. Particularly if the liquid level of the thus-pressurised system is on the sample holder side of the valve 384 instead of on the syringe side, the valve may also be closed to maintain the elevated pressure without having to rely on the syringe and clamp. [0160] H—Reducing the sample liquid pressure from the elevated pressure to the operating pressure by relaxing the clamp and retracting the piston from the syringe gently to the desired position associated with the operating pressure. This may mean retracting the piston to the position at the end of step C above.

[0161] The disclosure is not restricted to the above described embodiments which can be varied in a number of ways within the scope of the claims.

[0162] For instance another compressor than a syringe with or without a clamp may be used. The compressor may be computer-controlled. Also, the sample holder may be designed and/or used for other types of microfluidic experiments and/or purposes than acoustic force measurements and/or optical tweezers experiments.

[0163] Elements and aspects discussed for or in relation with a particular embodiment may be suitably combined with elements and aspects of other embodiments, unless explicitly stated otherwise.