METHOD AND SYSTEM FOR STUDYING OBJECTS, IN PARTICULAR BIOLOGICAL CELLS

Abstract

Systems and methods for manipulating and/or investigating objects, in particular biological objects such as cellular bodies, in a sample holder are provided. The sample holder comprises a holding space for holding a sample comprising one or more objects in a fluid medium. An acoustic wave generator is connected with the sample holder to generate an acoustic wave in the holding space exerting a force on at least part of the sample. Fluid flows through microfluidic channels in the sample holder and acoustic waves are controlled.

Claims

1. A method of manipulating and/or investigating objects, comprising: providing a sample holder comprising a holding space for holding a fluid medium; providing a sample comprising one or more objects in a fluid medium in the holding space; generating an acoustic wave in the holding space exerting a force on the one or more objects of the sample in the holding space; providing a sample fluid flow through the holding space; and providing a sheath flow adjacent the sample fluid flow and controlling, using the sheath flow, at least one of a size, location and/or a path of the sample flow in at least part of the holding space.

2. The method according to claim 1, comprising: wherein providing a sample comprising one or more objects in a fluid medium in the holding space comprises providing the sample into the holding space along a first flow path in the sample holder, and wherein the method further comprises providing one or more second fluid flows in the sample holder along a second flow path, and in case of more second fluid flows each along a different respective second flow path, wherein at least part of the one or more second flow paths and the first flow path pass through a common channel and/or the holding space.

3. The method according to claim 2, comprising providing one or more second fluid flows through the holding space.

4. The method according to claim 2, wherein one or more parameters of the acoustic wave are adjustable, the one or more parameters of the acoustic wave being selected from application, power, amplitude, wavelength, frequency and variations thereof, and wherein the method comprises providing the second fluid flow or at least one of the one or more second fluid flows, respectively, in dependence of at least one of the one or more parameters of the acoustic wave.

5. The method according to claim 1, comprising: wherein providing a sample comprising one or more objects in a fluid medium in the holding space comprises providing the sample into the holding space along a first flow path in the sample holder through a first channel, and wherein the method further comprises providing an interaction substance source in a reservoir in the sample holder, establishing a fluid contact between the source and the sample space and providing at least some of the interaction substance to the objects by diffusion via a diffusion channel between the reservoir and the holding space.

6. The method according to claim 1, wherein in particular the one or more objects comprise biological objects; wherein the method further comprises providing the holding space with a functionalised wall surface portion to be contacted by the sample and wherein the sample is in contact with the functionalised wall surface portion during at least part of the step of generating the acoustic wave.

7. The method according to claim 1, comprising detecting one or more signals indicative of adhesion and/or detachment of at least one of the one or more objects to/from at least part of the sample holder.

8. The method according to claim 7, comprising detecting the one or more signals indicative of adhesion and/or detachment of at least one of the one or more objects to/from at least part of the sample holder as a function of at least one parameter of the acoustic wave selected from duration of application, power, amplitude and frequency.

9. The methods according to claim 7, comprising separating at least one object from the one or more objects, based on at least one parameter of the acoustic wave.

10. A system for manipulating and/or investigating objects, comprising a sample holder comprising a holding space for holding a sample comprising one or more objects in a fluid medium, and an acoustic wave generator connected with the sample holder to generate an acoustic wave in the holding space exerting a force on at least part of the sample, wherein the sample holder comprises a first microfluidic sample channel provided with a fluid inlet and a fluid outlet for generating a sample fluid flow of the fluid medium through the holding space and a first microfluidic channel portion for generating a first sheath flow portion in at least part of the first channel and the holding space adjacent the sample flow, and a second microfluidic channel portion for generating a second sheath flow portion in at least part of the first channel and the holding space adjacent the sample flow opposite from the first sheath flow.

11. The system according to claim 10, comprising wherein the sample holder comprises a first microfluidic channel provided with a first fluid inlet and a first fluid outlet and defining a first flow path in the sample holder from the first inlet to the first outlet, and a second microfluidic channel comprising a second fluid inlet and a second fluid outlet and defining a second flow path in the sample holder from the second inlet to the second outlet, such that at least part of the first and second flow paths pass through a common channel and/or the holding space.

12. The system according to claim 11, wherein the sample holder comprises a plurality of microfluidic channels provided with an inlet and/or an outlet, each of the plurality of microfluidic channels defining at least part of a flow path in the sample holder from a respective inlet to a respective outlet, such that each respective separate flow path crosses and/or intersects the first flow path.

13. The system according to claim 11, wherein the sample holder comprises a plurality of microfluidic channels provided with an inlet and/or an outlet, each of the plurality of microfluidic channels defining at least part of a separate flow path in the sample holder from the respective inlet to the holding space and/or from the holding space to the respective outlet, such that each respective separate flow path crosses and/or intersects the first flow path in the holding space.

14. The system according to claim 10, wherein the system comprises a controller connected with the acoustic wave generator and being configured to control one or more parameters of the acoustic wave, and wherein the system comprises a fluid flow controller connected to the inlets and outlets of the sample holder and being configured for providing a fluid flow along each of the respective flow paths.

15. The system according to claim 10, the system comprising wherein the sample holder comprises at least one nutrient and/or interaction substance reservoir, at least one a diffusion channel connecting the reservoir to the holding space separate from the first channel.

16. The system according to claim 10, the system comprising wherein the sample holder comprises a first microfluidic channel provided with a fluid inlet and a fluid outlet for generating a sample fluid flow of the fluid medium through the holding space and wherein the sample holder comprises one or more microfluidic channels provided with at least one of an inlet and an outlet and being connected with the first channel remote from the holding space for providing a fluid flow in and/or through at least part of the first channel offset from and not through the holding space.

17. A method of manipulating and/or investigating objects, comprising: providing a sample holder comprising a holding space for holding a fluid medium; providing a sample comprising one or more objects in a fluid medium in the holding space; generating an acoustic wave in the holding space exerting a force on the one or more objects of the sample in the holding space; wherein providing a sample comprising one or more objects in a fluid medium in the holding space comprises providing the sample into the holding space along a first flow path in the sample holder, and providing one or more second fluid flows in the sample holder along a second flow path, and in case of more second fluid flows each along a different respective second flow path, wherein at least part of the one or more second flow paths and the first flow path pass through a common channel and/or the holding space.

18. The method according to claim 17, comprising providing plural second fluid flows through the holding space, each along a different respective second flow path, wherein each of the respective different second flow paths pass through the holding space, in particular crossing and/or intersecting the first flow path in the holding space.

19. The method according to claim 17, wherein one or more parameters of the acoustic wave are adjustable, the one or more parameters of the acoustic wave being selected from application, power, amplitude, wavelength, frequency and variations thereof, and wherein the method comprises providing the second fluid flow or at least one of the one or more second fluid flows, respectively, in dependence of at least one of the one or more parameters of the acoustic wave.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] 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.

[0078] FIG. 1 is a schematic drawing of an embodiment of a manipulation system;

[0079] FIG. 2 is a schematic drawing of a sample holder for the system of FIG. 1;

[0080] FIG. 2A is a schematic detail of FIG. 2 as indicated;

[0081] FIGS. 3-11 show a workflow for acoustic force measurements;

[0082] FIG. 12 shows an embodiment of a sample holder for use in the system of FIG. 1;

[0083] FIG. 13-14 show an embodiment for flow focusing using sheath flows;

[0084] FIG. 15-19 show embodiments of sample holders and workflows;

[0085] FIGS. 20-22 show embodiments of a sample holder and workflow steps in cross section;

[0086] FIGS. 23-24 show a cross section of part of a holding space during a method disclosed herein.

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 molded 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 an embodiment of a manipulation system 1 in accordance with the present concepts, 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 sample holder 3 comprising a holding space 5 for holding a sample 7 comprising one or more objects like biological cellular bodies 9, in a fluid medium 11 as exemplary particles of interest. It is noted that also, or alternatively, other types of particles like microspheres could be used, possibly attached to biological cellular bodies 9. Microspheres of polymeric and/or glass material (hollow and/or solid) may be suitable objects; such microspheres may be coated wholly or in part with any suitable coating and/or primer. The fluid preferably is a liquid or a gel. 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 exerting a force on the sample 7 and cellular bodies 9 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 functionalised wall surface portion 17 to be contacted, in use, by part of the sample 7. Here, the functionalised 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 layer in between (not shown). As explained in more detail below, interaction of the cellular bodies 9 (and/or other objects) with the cellular bodies 10 may be studied with the systems and methods. A further wall, e.g. opposite wall 16, may also or alternatively be provided with a (further) functionalised 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/flow sensors, pressure regulators, etc. in order to facilitate control over fluid flows. This enables also specific flow protocols to be incorporated into the experimental protocols and/or enables automation of flow based on detection of certain events by the system. Such events may comprise changes in at least part of a sample, e.g. changes in position and/or movement of one or more objects in the sample such as (induced by and/or otherwise associated with) adhesion and/or detachment of the one or more objects to/from the functionalised wall surface.

[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 further optics (not shown) to the camera 23. The objective 21 and the camera 23 may be integrated. In an embodiment, two or more optical detection tools, e.g. with different magnifications, may be used simultaneously for detection of sample light 33, e.g. using a beam splitter. The computer may also be connected to the light source 27 e.g. in order to synchronize the light source with the camera.

[0094] In another embodiment, not shown but discussed in detail in WO 2014/200341, the system comprises a partially reflective reflector and light emitted from the light source is directed via the reflector through an objective and through the sample, and light from the sample is reflected back into the objective, passing through the partially reflective reflector and directed into a camera possibly via intervening optics. Further embodiments may be apparent to the reader.

[0095] 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.

[0096] Some optical elements in the system 1 may be at least one of partly reflective, dichroic (having a wavelength specific reflectivity, e.g. having a high reflectivity for one wavelength and high transmissivity for another wavelength), polarisation selective and otherwise suitable for the shown setup. Further optical elements e.g. lenses, prisms, polarizers, diaphragms, reflectors etc. may be provided, e.g. to configure the system 1 for specific types of microscopy.

[0097] 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 an enclosed holding space 5 in cross section.

[0098] The sample holder 3 preferably is a substantially planar device, more preferably a microfluidic device of the type commonly referred to as a lab-on-a-chip. At least part of the sample holder may be formed by a single piece of material with a channel 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 a holding space 5 is formed in which the fluid sample 7 is 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 acoustical 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.

[0099] As shown in FIG. 2, the sample holder 3 is connected to an optional fluid flow system 35 for introducing fluid into the holding space 5 of the sample holder 3 and/or removing fluid from the holding space 5, e.g. for flowing fluid through the channel 4 and holding space 5 (see arrows in FIG. 2), 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 fluids and outlet conduits 39 for removing one or more fluids, 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 preferable 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 functionalised 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 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 W away from the surface 17, generating a local maximum force F on the bodies 9 at or near the surface towards the node. Thus, as explained in detail in WO 2018/083193, application of the signal may serve to probe adhesion/detachment of the bodies 9 to the surface and/or any functionalised layer on it in dependence of the strength of the force.

[0102] The optimal force generation may be achieved by designing optimizing the acoustic cavity parameters and the frequency/wavelength of the acoustic wave such as to create a maximum pressure gradient at the functionalized wall surface (e.g. by ensuring the distance from the wall surface to the acoustic node is ¼ wavelength).

[0103] 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, and/or by the location of the acoustic wave generator 13 and/or 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 (FIG. 1).

[0104] FIGS. 4 and 5 indicate loading the channel 4 and the holding space 5 with a sample fluid and target cells 10 via the inlet 41, e.g. using the conduit 38 and/or another fluid supply 47 such as a pipette. The target cells 10 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 functionalised wall surface portion 17 is formed, see FIGS. 5 and 6 cf. FIG. 2k Formation of the functionalised wall surface portion 17 may comprise further suitable steps such as giving the target cells time to attach to the surface and grow into a monolayer. Also, a functionalised wall surface portion 17 may (be formed to) comprise different portions and/or portions with different properties.

[0105] The functionalised wall surface portion 17 tends to be distributed over the channel 4 beyond the holding space 5, and may include conduits 38 and/or 39 if used. The loaded cells 10 may be incubated and/or cultured in the channel 4. However, it has been found that loaded cells 10 may be (negatively) affected if the medium is not refreshed during incubation/culturing. Also, a fluid flow through the channel 4 and holding space 5 may cause motion stress on the cells 10. In particular, the cells may at least partly detach from the surface and/or (attempt to) assume a spherical shape to minimise surface area and/or reduce contact to other surfaces, including neighbouring cells. This may affect (further) experimental results and/or cause deformation and/or structuring of the layer 17. See also FIGS. 23-24 discussed below. Flow rates over a few tens of nanoliters per minute in microfluidic channels and/or shear stress levels over 1 mPa may need to be avoided in order not to negatively affect the cells,

[0106] FIG. 6 indicates, some later time, loading a sample comprising objects 9 such as effector cells 9 or other cellular bodies in a sample fluid 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.

[0107] FIG. 7 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) fluid flow, which may (further) affect at least some of the target cells 10 and/or the effector cells 9.

[0108] FIG. 8 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 functionalised wall surface 17 towards the node in the sample fluid (indicated as cells 9′ with a lighter colour), while cells 9 that are stronger bound to the functionalised wall surface 17 may remain adhered (cf. FIG. 2A). Thus, a separation is made between different types of effector cells 9 based on adhesion characteristics; unbound or loosely bound vs. strongly bound.

[0109] FIG. 9 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, which later-detached cells may be separately collected.

[0110] Note that between successive and/or stronger acoustic wave periods in the holding space 5 one or more sample modifications may have been provided and/or further instances may have been allowed to happen and/or caused to happen as part of a measurement, therapy and/or experiment, e.g. one or more of settling, aging, reaction, interaction, cultivation, heating, cooling, irradiation, and/or other (bio-)physical and/or (bio-)chemical processes.

[0111] As will be appreciated from FIG. 9, the cells 9 detached in the holding space 5 will have to travel through lead-out section 4B of the channel 4 between the holding space 5 and the outlet 43. Thus, the sample portion of sample fluid and cells 9 may become contaminated and/or entrained cells 9 may get lost, e.g. further interaction between the different types of cells 9, 10 may occur which may be undesired and/or cells 9 may (otherwise) get stuck onto the cells 10 and/or cells 10 may become detached and flush out together with the flushing fluid and desired cells 9.

[0112] Note that the terms “inlet” and “outlet” may generally relate to the direction of a fluid flow through the respective structure, 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. 8-9, 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 fluid.

[0113] Moreover, as FIG. 10 indicates, it has been found that, in practice, the acoustic force may not be distributed evenly over the holding space but that local force variations may occur. For example, the acoustic force at the center of the holding space may be higher than at the edges of the holding space. Therefore not all cells in the holding space may be experiencing the same force at the same time. Moreover, cells 9 with different detachment characteristics may be collected as one batch after flushing out (cf. FIG. 9). Also or alternatively, flow effects in the sample holder channel 4 and/or holding space 5 may cause uneven distribution of functionalisation moieties for establishing the functionalised wall surface portion 17 and/or uneven distribution of the objects 9 and/or uneven flushing out of detached cells 9 may occur. Such imperfections should be reduced and/or prevented; the more specific the force and/or time characteristics of the detachment are and/or the better one or more of these are known and/or controlled, the more specific the selection of objects 9 may be and the more precise effects may be studied and/or used.

[0114] FIG. 11 shows an option for selecting different portions of objects 9 studied with the sample holder 3: a splitter 51 is connected to (the outlet 43) of the sample holder 3, e.g. using tubing 52. The splitter 51 comprises a plurality of outlets 53 for selected batches of objects 9 removed from the sample holder 3. The splitter 51 may comprise one or more valves (not shown) and/or more or less outlets 53.

[0115] FIG. 12 shows an embodiment of a sample holder 3A wherein instead of the splitter 51 of FIG. 11, plural fluid outlets 43A-43C may also be integrated into the sample holder 3, connected with the channel 4 and the holding space 5 via individual microfluidic channels 55A-55B. This facilitates reduction of channel lengths so that smaller amounts of (possibly dangerous, delicate and/or expensive) fluids may be used and sample portions may be better selectable, e.g. by reduction or prevention of dispersion.

[0116] FIG. 13 shows a sample holder 3B for microfluidic control. The sample holder 3B comprises a holding space 5 for holding a sample comprising one or more objects 9 in a fluid medium, and an acoustic wave generator 13 connected with the sample holder 3 to generate an acoustic wave in the holding space 5 exerting a force on the sample. Further, the sample holder 3B comprises a first microfluidic channel 4 provided with a fluid inlet 41A and a fluid outlet 43A, here being connected to the microfluidic channel 4 via channels 57A, 55A for generating a sample fluid flow 59 of the fluid medium comprising through the holding space 5 (black arrow). Here, the fluid medium may comprise sample objects like cellular bodies 9. The sample holder 3B further comprises a first microfluidic channel 57B for generating a first sheath flow portion 61A (white arrow) in at least part of the first channel 4 and the holding space 5 adjacent the sample flow 59, and a second microfluidic channel 57C for generating a second sheath flow portion 61B in at least part of the first channel 4 and the holding space 5 adjacent the sample flow 59 opposite from the first sheath flow 61A (white arrow). The sheath flows may comprise and/or consist essentially of the sample liquid but without sample objects 9.

[0117] By controlling the absolute and relative flow strengths flux of each of the flows 59, 61A, 61B while maintaining all flows 59, 61A, 61B in the laminar flow regime as a whole and with respect to each other, the flows 59, 61A, 61B substantially do not mix in the channel 4 and the holding space 5. The control may comprise one or more of fluid pressure, fluid flow volume and flow velocity, possibly controlled using one or more controllable valves, sources, buffer reservoirs and/or pressurizers, etc. By flowing sheath fluids adjacent a sample fluid in the first channel in a laminar flow, the fluids will substantially remain unmixed and by adjustment of flow rates of the three flows with respect to each other (in particular the first and second sheath flows with respect to each other and with respect to the sample flow) a volume, position and direction of the sample flow through the first channel and/or the holding space can be controlled. The adjustment of the flow for any of the flows 59, 61A, 61B may range from no flow to fully filling up the channel 4 and/or holding space 5 with a single flow, effectively blocking the channel for further contributions of the other ones of the flows 59, 61A, 61B. Thus, the sample flow 59 may be controlled with respect to at least one of a size, location and/or a path of the sample flow 59 in at least part of the holding space 5. In such way an interaction position (or interaction region) in the holding space 5 may be selected, e.g. with respect to at least part of the functionalised wall surface 17 and/or a specific location of the acoustic force. During and/or after experiments this may also serve for flushing and/or collecting sample portions from specific locations of/from the holding 5.

[0118] From comparison of FIGS. 12 and 13 may be seen that one sample holder design may be used for both methods, also in combination, such as e.g. introduction of a sample portion as presented in (relation to) FIG. 13, and selection of different portions of objects 9 as presented in (relation to) FIG. 12 by suitably (re) connecting different sources and/or receptacles and reversing flow directions in at least part of the channels.

[0119] FIG. 14 shows a further sample holder 3C for microfluidic control, comprising both plural fluid outlets 43A-43C connected with the channel 4 and the holding space 5 via microfluidic sheath flow channels 55A-55B and plural fluid inlets 41A-41C connected with the channel 4 and the holding space 5 via microfluidic sheath flow channels 57A-57B. Thus, a first microfluidic sheath flow channel is provided with a first fluid inlet 41B and a first fluid outlet 43B and defining a first sheath flow path 41B-57B-4A-5-4B-55B-43B in the sample holder from the first inlet 41B to the first outlet 43B, and a second microfluidic sheath flow channel is provided with a second fluid inlet 410 and a first fluid outlet 430 and defining a second sheath flow path 410-57B-4A-5-4B-55C-43C in the sample holder 3C from the first inlet 41C to the first outlet 43C. Such sample holder 3C may be seen as a combination of the sample holders 3A and 3B of FIGS. 12 and 13. Thus, the sample flow and sheath flows 59, 61A-61B may be controlled more accurately. Also or alternatively, different sample portions may be flushed to selective ones of the outlets 43A-43C, Other designs of sample holders may be provided, e.g. comprising more, less and/or differently connected microfluidic sheath flow channels for generating sheath flows and/or for defining (sheath) flow paths.

[0120] FIGS. 15-19 show a sample holder 3D reminiscent of the sample holders 3-3C. This sample holder 3D comprises a holding space 5D for holding a sample comprising one or more objects e.g. cellular bodies 9, 10, in a fluid medium, and an acoustic wave generator 13 to generate an acoustic wave in the holding space 50 exerting a force on at least part of the sample. The sample holder 3D comprises a first microfluidic channel 4 comprising channel portions 4A, 4B, being provided with a first fluid inlet 41 and a first fluid outlet 43 and defining a first flow path FP4 (41-4A-5D-4B-43) in the sample holder 3D from the first inlet 41 to the first outlet 43. The sample holder 3D further comprises a second microfluidic channel 63 comprising first and second channel portions 63A, 63B, and being provided with second fluid inlet 65 and a second fluid outlet 67 and defining a second flow path FP63 (65-63A-5D-63B-67) in the sample holder 3D from the second inlet 65 to the second outlet 67. The first and second flow paths FP4, FP63 extend perpendicular to each other and intersect each other in the holding space 5D, but note that other angles than perpendicular may also be provided.

[0121] FIGS. 15-19 indicate an exemplary method of manipulating and investigating objects in the sample holder 3D.

[0122] FIG. 15-16 indicate loading the holding space 5D with a sample fluid and target cells 10 via the inlet 41, cf. FIGS. 4-5. The target cells 10 are distributed via the first fluid path FP4 over the channel 4 and the holding space 50 and a functionalised wall surface portion 17 is formed.

[0123] FIGS. 17-18 indicate subsequent loading of a sample comprising effector cells 9 and/or other objects (not shown) in a sample fluid into the holding space 5D from a supply 49 via the first fluid path FP4 over the channel 4. The effector cells 9 and target cells 10 may be left to interact as discussed above. At a desired time an acoustic wave is generated in the holding space 5D by the acoustic wave generator 13, exerting a force on the cellular bodies 9 and/or other objects in at least part of the sample in the holding space 5D, with which at least some of the cellular objects 9 are suspended in the sample fluid and/or detached from the functionalised surface portion 17.

[0124] FIG. 19 shows providing a second fluid flow through the holding space 5D along a second flow path FP63 (indicated by the arrows). This may be done during and/or after generating the acoustic wave in the holding space 5D, and/or otherwise in dependence of at least one of the one or more parameter of the acoustic wave and/or in dependence to an observed parameter in the holding space. Since the second flow path FP63 only intersects the first flow path FB4 in the holding space 5D, the sample portion (in particular detached cells) flushed out of the holding space 5D are passed through second flow channel 63 without further interaction with cells in the second channel portion 4B of the first channel 4, and/or without being otherwise contaminated or affected by sample portions in that channel (portion). Thus, preparing of a sample and obtaining results from the sample may be largely decoupled.

[0125] FIG. 20 shows an embodiment of a sample holder 3E comprising a holding space 5E and comprising plurality of microfluidic channels 69A-69C provided with an outlet 71A-71C. Each of the plurality of microfluidic channels 69A-69C defines a separate flow path in the sample holder 3E from the holding space 5E to the respective outlet 71A-71C, each starting from the inlet 65 such that each respective separate flow path 65-63A-5E-69A/69B/69C-71A/71B/71C at least partly intersects the first flow path FP4 in the holding space 5E. Thus, similar to FIG. 12, plural fluid outlets 71A-71C are directly accessible for fluid flows from the holding space 5E as exit ports for selecting and/or sorting sample portions. Also or alternatively, microfluidic channels 69A-69D and the outlets 71A-71C may be used for microfluidic sheath flows for controlling at least part of a sample flow and/or a sorting/selection flow from one or more particular portions of the holding space 5E. E.g., a fluid flow from a supply 68 may flush out detached cellular objects 9 to channel 69A and outlet 71A.

[0126] In yet another embodiment, not shown, the sample holder 3E of FIG. 20 may be used in reverse to the description of FIG. 20, such that the ports 71A-71C and channels 69A-69C are used as fluid inlets for introducing a fluid to the holding space 5E, possibly a sample fluid, cf. FIG. 12 relative to FIG. 13. In yet a further embodiment, not shown, the sample holder comprises a plurality of microfluidic channels provided with an outlet on one side of the first channel and holding space, as in FIG. 20, as well as a similar—not necessarily identical—set of microfluidic channels provided with an inlet on an opposite side of the first channel as just before described. In such embodiment, microfluidic flow control may be further improved, comparable to (the discussion of) FIG. 14 (or the discussion with respect to FIGS. 12-14 as a whole).

[0127] Also, combinations of embodiments of FIGS. 12-14 with FIGS. 15-19, FIG. or the embodiments described in the preceding paragraph could be provided and/or used.

[0128] FIG. 21A shows an embodiment, combinable with any other embodiment, of a sample holder 3F provided with an acoustic wave generator 13. The sample holder 3F comprises two microfluidic channels 73A, 73B each provided with an inlet or outlet 74A, 74B or, respectively, and being connected with the first channel 4 remote from the holding space 5 for providing a fluid flow in and/or through at least part of the first channel 4 offset from and not through the holding space 5. Thus, at least part of the first channel 4 may be flushed, e.g. by providing a fluid flow along a flow path 74A (and/or 74B)-73A (and/or 47B)-4B-43 and/or a flow path 74A-73A-4B-73B-74B and/or along such flow path in an opposite flow direction. This may also provide a way to provide nutrients and/or interaction moieties for either target cells 10 or cellular bodies 9 by diffusion from channel portion 4B to holding space 5 without disturbing the cells in the by any significant flow and/or shear stress.

[0129] Alternatively inlets and outlets may be placed on opposite sides of holding space 5 as shown for a sample holder 3G in FIG. 21B. Such embodiment facilitates a fluid flow along a flow path 74A-73A-4A-41 (or in opposite direction) and fluid flow along a flow path 74B 73B-4B-43 (or in opposite direction), possibly without net flow through the holding space 5. Clearly, a first flow path 41-4A-5-4B-43 (or in opposite direction) and a second flow path 74A-73A-4A-5-4B-73B-74B (or in opposite direction) cross and/or intersect in part of the channel 4 and in the holding space 5.

[0130] FIG. 21C shows an embodiment functionally substantially identical to sample holder 3G of FIG. 21B, wherein, compared to FIG. 21B, the flow paths channels 73A, 73B and inlets/outlets 74A, 74B arranged on the same side of the channel 4. Thus, a second flow path 74A-73A-4A-5-4B-73B-74B is generally U-shaped. Such embodiment could facilitate mounting and/or use in the system of FIG. 1 since connections to inlets/outlets 74A, 74B could be arranged on a common side. Channel 4 could also be provided with a U-shape and/or all connections could be arranged on a common side.

[0131] Placing the second flow path channels 73A, 73B outside of the holding space may serve to minimize impact of the side channels on the acoustic wave and/or associated distribution of acoustic forces.

[0132] Hg. 22 shows another embodiment of a sample holder 3H provided with an acoustic wave generator 13. The sample holder 3H comprises a holding space 5G for holding a sample comprising one or more objects in a fluid medium. The acoustic wave generator 13 is connected with the sample holder 3H to generate an acoustic wave in the holding space 5G exerting a force on at least part of the sample. The sample holder 3H comprises an inlet 41, a channel 4 having first and second channel portions 4A, 4B, and an outlet 43 for providing a sample comprising one or more objects to the holding space 5G as discussed in any embodiment before. The sample holder 3H comprises two interaction substance reservoirs 75 adjacent the holding space 5G. A plurality of diffusion channels 77 arranged on opposite sides of the holding space 5G connect each reservoir 75 to the holding space 5G separate from the first channel 4. Thus, any interaction substance, e.g. nutrients and/or interaction moieties for a sample containing biological matter, may be distributed along the holding space 5G so that undesired differences and/or gradients of the interaction substance in the holding space may be reduced or prevented. The number and/or distribution of the diffusion channels 77 and/or the shape and/or size of the reservoir may be provided with respect to one or more properties of the sample and/or the interaction substances. Also or alternatively, as in any embodiment, the acoustic wave generator 13 may be shaped and/or localised differently.

[0133] The diffusion channels 77 may be formed to filter substances between a reservoir 75 and the holding space 5G and/or to substantially decouple the reservoir 75 and the holding space 5G from flow and/or pressure differences in one of the reservoir 75 and the holding space 5G when manipulating (e.g. filling, changing, emptying, etc.) the other one of the reservoir 75 and the holding space 5G, and/or from effects of the acoustic wave, e.g. by providing at least part of the diffusion channels 77 with an appropriate size and/or pattern and/or other shape (e.g. bend).

[0134] In the embodiment shown in FIG. 22 the interaction reservoirs 75 are connected via channels 79 to an inlet 81 and via channels 83 to a common outlet 43. A common inlet assists providing a homogeneous distribution of the substance over the reservoirs 75 connected to the inlet with respect to one or more of composition, amount, pressure, flow velocity etc. However, one or more reservoirs 75 may be provided with independent inlets and/or outlets. Also or alternatively, one or more reservoirs 75 may be provided with one or more of separate channels and/or one or more separate inlets and/or outlets for independent control of a substance in the respective reservoir(s) 75.

[0135] Also or alternatively, the sample holder 3H may be used similar to the sample holder 3F of FIG. 21, i.e. the outlet 43 and a portion of the second channel portion 4B may be flushed by flushing a fluid via the inlet 81, channels 79 and 83 including the reservoirs 75 and outlet 43. Then objects and/or a sample fluid from a sample in the holding space 5G may be flushed to the outlet 43 in the same fashion as (described for) the embodiments of FIGS. 3-11 with little or no contamination of remaining substances in the outlet and possibly associated conduits. And also without disturbing the sample in the holding space. This may improve for example the ability to collect a batch of cells (e.g. a batch of cells which detached from a functionalized wall surface portion due to a specific detachment force) into a specific collection batch without contamination by a next batch of cells detaching at a higher acoustic force. i.e. if a batch of cells detaches inlet 41 can be temporarily opened and a small amount of flow in channel 4 and holding space 5G can be used to flush the batch of cells to the junction between channels 4B and 83. After this the inlet 41 can be closed and flow from inlet 81 can be used to flush the batch of cells from the junction all the way to the collection area where for example a splitter similar to splitter 51 in FIG. 11 can be used to send the batch to a specific collection sample. Then the flow from inlet 81 can be closed, the force can be increased and the next batch of cells can be gently flown to the junction after which this batch can be collected according to the same process.

[0136] Note that in this embodiment, as in any other embodiment, the words “inlet” and, respectively “outlet” are used primarily for ease of reference and may refer only to the sample holder in operation and with respect to a particular fluid flow direction, possibly governed by outside control systems, whereas the sample holder itself does not determine, define or suggest any particular in- and/or outflow direction. E.g., the connections 41, 43, 81 in the sample holder 3H may in practice be used as an inlet, as an outlet or as both as an inlet and an outlet subsequently, possibly within one experiment/experimental sequence; see also the discussions regarding FIGS. 12-13 and FIG. 20.

[0137] As discussed in relation to FIGS. 5 and 6 above, FIG. 23 indicates a cross section of part of a holding space of a sample holder as discussed herein comprising a first wall 15 and a second wall 16 defining a cavity 85 of the holding space. The (cavity 85 of the) holding space is being provided with a sample fluid 11 and a functionalised wall surface portion 17 on the basis of cellular bodies 10, cf. FIG. 2A.

[0138] FIG. 24 indicates the cross section of the sample holder of Hg. 23 subject to a sample fluid flow SFF of the fluid 11. In response to the sample fluid flow SFF the is cells 10 have taken up a more globular shape interrupting the functionalised wall surface portion 17.

[0139] 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.

[0140] Various embodiments of methods and/or method steps may be implemented as a program product for use with a computer system, where the program(s) of the program product define functions of the embodiments (including the methods described herein). E.g., controlling operation of one or more of the acoustic wave generator, one or more valves, one or more pumps, temperature control devices, cameras, etc. In one embodiment, the program(s) can be contained on a variety of non-transitory computer-readable storage media, where, as used herein, the expression “non-transitory computer readable storage media” comprises all computer-readable media, with the sole exception being a transitory, propagating signal. In another embodiment, the program(s) can be contained on a variety of transitory computer-readable storage media. Illustrative computer-readable storage media include, but are not limited to: (i) non-writable storage media (e.g., read-only memory devices within a computer such as CD-ROM disks readable by a CD-ROM drive, ROM chips or any type of solid-state non-volatile semiconductor memory) on which information is permanently stored; and (ii) writable storage media (e.g., flash memory, floppy disks within a diskette drive or hard-disk drive or any type of solid-state random-access semiconductor memory) on which alterable information is stored.

[0141] 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.