Resonance matched and power compensated actuator device for acoustic particle manipulation

Abstract

The present disclosure relates to an ultrasonic actuation device arranged for providing an ultrasonic actuation to at least one resonance structure of at least one fluidic device, the actuation device comprising: a planar actuation frame arranged for accommodating the fluidic device in a fluidic device area; and an actuation arrangement comprising at least one actuation source having a resonance plane connected to the actuation frame, wherein the at least one actuation source is arranged to be, in use, laterally coupled to the fluidic device.

Description

[0001] Resonance matched and power compensated actuator device for acoustic particle manipulation

[0002] The present invention relates to ultrasonic actuation devices, systems, and assemblies, and use thereof. Further, the invention relates to methods for actuation of devices, systems and assemblies.

[0003] Background

[0004] Cell aggregations, commonly known as 3D cell cultures, have emerged as valuable tools for researchers due to their ability to provide a more realistic representation of the in vivo environment compared to traditional 2D cell cultures. Numerous studies and reviews have highlighted significant genetic variations between cells cultured in 2D and 3D, particularly in the upregulation of three key categories: cell cycling, metabolism, and macromolecule turnover.

[0005] These differences contribute to enhanced cellular functions and proliferation, making 3D cell aggregations essential for studying tissue development, cell-cell interactions, cell differentiation, viability, migration, and tissue formation. As a result, 3D cell cultures have found applications in drug discovery and delivery, and are indispensable in cancer research, where they mimic the heterogeneous architecture and internal gradients present in solid tumors.

[0006] However, significant limitations exist in current spheroid formation techniques. One limitation is the restricted number of cells available for sample isolation, protein analysis, and nucleic acid extraction. Additionally, maintaining consistency and controlling variability within each spheroid remains challenging. To address these limitations, various techniques have been explored. For example, miniaturized devices have enabled precise cell handling and manipulation at relevant length scales.

[0007] Additionally, surface coatings with hydrophobic biomaterials have been developed to prevent cell adhesion and promote spheroid formation as cells aggregate. Another approach involves using spinner flask plates with conical bottoms to induce cell aggregation and spheroid formation.

[0008] Recent advancements have introduced new methods for forming spheroids, such as the approach proposed by Vanherberghen et al. Lab Chip, 10, 2727, 2010. This study demonstrated the application of ultrasonic particle manipulation, including focusing, separating, sorting, mixing, and patterning, in an open multi-well microplate for parallelized aggregation of particles, cells, or organisms at specific locations. This study achieved parallelization through a frequency modulation scheme, ensuring stable particle positioning over extended periods. Another example is US 2013 / 000420A, which discloses a method and a device for simultaneously merging suspended particles and / or cells in discrete microwells. These approaches highlight the potential of ultrasonic manipulation for advancing cell culture technologies.

[0009] The object of the present invention is to overcome the challenges of the prior art devices and methods, which are typically characterized by limitations such as significant heating, high energy demands, and restricted control over resonance frequencies, and inefficient focusing.

[0010] Summary

[0011] Devices of the prior art are characterized by multiple resonances averaged into a field, compelling all particles to converge at the center, while multiple wells enable parallel spheroid generation. However, the use of broadband actuation presents challenges in terms of significant heating, high demands on electrical drive systems (such as high voltage and power), and reliance on active cooling systems.

[0012] To address these limitations of the prior art devices and methods, the present inventors have realized how ultrasonic actuation devices may be arranged in order to efficiently provide ultrasonic actuation to fluidic devices, without cooling requirements and wherein the resonance frequency is independent of the fluidic device.

[0013] Therefore, in a first aspect, the present disclosure relates to an ultrasonic actuation device arranged for providing an ultrasonic actuation to at least one resonance structure of at least one fluidic device, the actuation device comprising:

[0014] • a planar actuation frame comprising a fluidic device area arranged for accommodating the fluidic device; and

[0015] • an actuation arrangement comprising at least one actuation source connected to the actuation frame, wherein the at least one actuation source does not overlap the fluidic device area, in a resonance direction of the actuation source. Thus, the present disclosure may relate to an ultrasonic actuation device for generating ultrasound to actuate a fluidic device, and wherein the actuation is arranged such that the main axial resonance does not include the fluidic device. Vibrations from the ultrasonic actuation device may instead be provided in a lateral arrangement to the fluidic device, allowing for lateral coupling of the fluidic device to the actuator source. This configuration results in increased design freedom, optical access, and ease of use.

[0016] The ultrasonic actuation device can allow for the formation of 3D cell cultures in a highly parallelized manner. The fluidic device may for example comprise a large number of wells, wherein each a 3D cell culture is formed during use. Further, the formed spheroids have a high degree of uniformity in size and shape, and the microenvironment is highly controllable and robust.

[0017] The ultrasonic actuation device can also provide for an energy-efficient actuation of the fluidic device, thereby negating the need for cooling systems. In addition, unlike devices of the prior art, the ultrasonic actuation device is compatible with microscopy setups, thus allowing for real-time observation by continuous imaging during long-term cell culturing, such as cell culturing during multiple days.

[0018] In contrast to the prior art, the presently disclosed devices and methods allow for dynamic monitoring of the developing 3D cell culture, and can produce spheroids with more uniform sizes and shapes, while not being dependent on costly or complex power amplifiers or cooling systems.

[0019] The presently disclosed ultrasonic actuation device typically comprises an actuation arrangement comprising at least one actuation source, for example a piezoelectric transducer. The actuation sources may, each, be bonded to the planar actuation frame.

[0020] In one embodiment of the presently disclosed ultrasonic actuation device, the actuation arrangement is adapted to fit onto the actuation frame, the actuation arrangement may comprise a plurality of actuation sources, and wherein the actuation sources are sized and shaped such that they are confined onto the actuation frame, such as wherein the actuation arrangement does not overlap with the fluidic device area. Furthermore, the actuation source(s) may be provided with non-parallel walls in the lateral dimensions (perpendicular to the plane of the planar actuation frame) to limit unwanted oscillations.

[0021] Also, in one embodiment of the presently disclosed ultrasonic actuation device, the actuation arrangement comprises several (for instance four) actuation sources, such as piezoelectric transducers. The actuation sources may be distributed over the actuation frame, such as symmetrically over the actuation frame. In this way, the uniformity of the actuation is improved, such that each resonance structure of the fluidic device, for example a microwell, is provided with a more similar acoustic intensity profile.

[0022] The fluidic device typically comprises a resonance structure. In one embodiment of the present disclosure, the fluidic device comprises a plurality of resonance structures, for example a plurality of discrete microwells. Each of said one or more resonance structures may define a confinement that is typically arranged to comprise particles and / or cells during use. Each confinement may be defined by one or more surfaces of the resonance structure. Said surfaces of the resonance structure may for example comprise a solid material such as glass or plastics. Each of the one or more resonance structures typically comprises at least one opening, for example wherein said opening is arranged such that liquid can be introduced into the resonance structure and / or removed from the resonance structure.

[0023] Further, each of the one or more resonance structures may comprise a plurality of openings. For example, the resonance structure may be arranged such that the confinement is a through-hole or part thereof. Hence, each resonance structure may comprise at least two openings. The confinement of the resonance structure may for example be arranged as an opening, passage, and / or through-hole through a solid material, such as through a planar structure. In one example, the resonance structure is arranged to, during use, retain a liquid and / or sample comprising particles and / or cells, at least partly by surface tension.

[0024] In other examples, the resonance structures may be microwells that each is defined by a plurality of surfaces. A conventional microwell typically has four side surfaces, a bottom surface, wherein the bottom surface is opposite to an opening arranged for introduction and / or removal of the liquid and / or sample. Thus, the resonance structures may be confinements arranged to retain a liquid solely by their shape, e.g. a well structure, and / or the resonance structures may be confinements arranged to retain a liquid by other means, such as by surface tension.

[0025] In one example, each resonance structure may comprise or consist of a lower surface and side surfaces. The resonance structure may thus form an integral part of the fluidic device.

[0026] The lower surface, during use, of the resonance structure may together with the side surfaces define a confinement, such as for containing a cell suspension. The resonance structure / confinement may have an opening opposite of the lower surface, such as at an upper part of the resonance structure. In other examples, as disclosed herein the resonance structure does not have a lower surface, but may be arranged to retain the liquid by other means, such as by surface tension between the liquid and the other surfaces of the resonance structure, such as the side walls.

[0027] Additionally, the resonance structures, such as the surfaces of the resonance structures, may be formed in any suitable substrate material, including silicon, metals, polymeric materials, or glass. In a preferred embodiment of the present disclosure, the lower surface of the microwell is formed in an optically transparent material, such as glass. In this way visual inspection via optical microscopy is enabled. However, in other examples, the entire resonance structure may be formed in said optically transparent material.

[0028] In one embodiment of the present disclosure, the fluidic device comprises an array of microwells, such as a single array with 100 microwells (10 x10 array) or 576 microwells organized in 16 arrays of 6 x 6 microwells.

[0029] The presently disclosed ultrasonic actuation device is typically arranged such that, during use, the ultrasonic actuation device may further allow for simultaneously manipulation of suspended particles and / or cells in a plurality of discrete microwells of the fluidic device, for example as shown in Fig. 8. The presently disclosed ultrasonic actuation device may be arranged to provide a more efficient actuation of the fluidic device, by frequency matching of one or more actuation source(s), for example one or more actuation sources with a substantially narrow resonance peak, to the expected acoustic resonance of the individual microwells.

[0030] This allows for a more uniform excitation of the resonance structure of the fluidic device, in particular when actuating complex fluidic devices (e.g. fluidic devices with over 200 resonance structures, or even over 300 resonance structures).

[0031] The presently disclosed ultrasonic actuation device may have an improved power efficiency that is translated into improved throughput, flexibility, and lower cost, as well as an improved uniformity of resulting acoustic fields, such as across the resonance structure, such as in the plane of the planar actuation frame.

[0032] In a further aspect, the present disclosure relates to a fluidic system comprising an ultrasonic actuation device as disclosed herein and a fluidic device.

[0033] In yet a further aspect, the present disclosure relates to use of the fluidic system as disclosed herein, for production of 3D cell cultures, such as in wells of a fluidic device.

[0034] In even yet a further aspect, the present disclosure relates to a method for power- compensated calibration of an actuation arrangement comprising an actuation device, the actuation device comprising at least one actuation source arranged to be actuated at a constant voltage, the method comprising:

[0035] • measuring electrical impedance of the actuation device at a plurality of frequencies within a modulation bandwidth of the actuation device;

[0036] • determining, for each frequency, based on the measured impedances, the time step needed to obtain an equal predetermined energy input.

[0037] The presently disclosed method allows for adaptive frequency modulation which can enable automatic calibration. This produces a tailored actuation scheme for each of a plurality of fluidic devices, that is subsequently actuated by the actuation device.

[0038] This presently disclosed method is typically based on analyzing the electrical impedance at each single frequency within the modulation bandwidth. This information may be used to compensate for variations when sweeping the frequency. This results in more robust and uniform force fields with similar magnitudes between different micro-wells in each chip compared with actuation methods of the prior art.

[0039] Other important advantages with the presently disclosed method are that the fluidic devices can be inserted and removed from the actuating frame without a need for manual calibration of frequency and voltage before each experiment.

[0040] Description of Drawings

[0041] Fig 1 is a schematic illustration of a cross-section of one embodiment of the presently disclosed actuation device.

[0042] Fig 2 is a 3D schematic illustration of one embodiment of the presently disclosed actuation device.

[0043] Fig 3 is a schematic illustration of one embodiment of the presently disclosed actuation frame accommodating a fluidic device having multiple fluidic wells.

[0044] Fig 4 shows admittance and susceptance spectra graphs for two different ultrasonic actuation device configurations, with resonances observable as peaks in the admittance graphs.

[0045] Fig 5 shows admittance and susceptance spectra graphs for different ultrasonic actuation device configurations, with resonances observable as peaks in the admittance graphs.

[0046] Fig 6 shows a power spectrum for a matched and slanted actuation frame together with the timesteps used to construct a power-compensated sweep according to an embodiment of the present disclosure.

[0047] Fig 7 shows COMSOL Multi physics® simulations of the pressure and velocity fields produced at the fundamental resonance of a single micro-well, consisting of two orthogonal modes aligned diagonally in the well.

[0048] Fig 8 shows a comparison of the effect on the aggregate shape and position between single frequency actuation, linear sweep actuation and power compensated sweep actuation according to an embodiment of the present disclosure. Fig. 9 shows a quantification of the results obtained in Fig. 8.

[0049] Detailed description

[0050] Definitions

[0051] A fluidic device, also referred to herein as a resonance device, is, as used herein, any structure or apparatus designed to facilitate resonant wave interaction for the manipulation, alignment, or organization of particles, cells, fluids, or other materials within defined spaces. Such a device may include various structural features, such as channels, wells, chambers, partitions, or other structures, which serve as interaction points for resonant waves generated by an actuation source.

[0052] In one example, the resonance device may be arranged to temporarily or permanently accommodate (e.g., contain) a fluid and / or a liquid within one or more defined confinements. The resonance deice, or confinement thereof, may additionally, or alternatively, to said fluid and / or liquid, be arranged to accommodate cells and / or particles (i.e., any minute entities as disclosed herein), either temporarily or permanently. Thus, in specific examples of the disclosure, the resonance structure, e.g. said confinement, may be adapted to accommodate cells and / or particles without a liquid and / or a fluid, e.g. in a dry state.

[0053] The resonance device may, for example, comprise or consist of a multiwell plate or microchannels, enabling applications like cell culture, fluid analysis, or particle manipulation within a liquid medium. In other examples, the resonance device may be adapted for containing cells and / or particles in a dry state, without requiring a liquid environment.

[0054] In some embodiments of the present disclosure, one or more regions of the device may function as resonance structures. This may be areas adapted to allow acoustic interaction. This can allow for control over the behavior of the contained or positioned materials. In particular embodiments, the resonance structures are part of a confinement, configured to accommodate and / or contain cells, particles, and / or fluid.

[0055] The resonance device / fluidic device may be configured to be accommodated by an actuation frame, such as a planar actuation frame, e.g. of the ultrasonic actuation device. For example, the resonance device may be a planar structure that is sized or adapted to be positioned on the actuation frame, such as at a designated fluidic device area.

[0056] Resonance structure, as used herein, refers to a specific region or element within the resonance device that is designed to interact with ultrasonic waves at selected frequencies, facilitating precise manipulation, alignment, or organization of materials within or near the structure. These structures may be configured to enhance acoustic effects, such as the alignment, aggregation, or separation of particles and / or cells.

[0057] Confinement, as used herein, refers to a defined space or compartment within a resonance device. Said confinement may be adapted to, temporarily or permanently, accommodate fluids, particles, and / or cells. The confinements may, for example, include one or more structural features, such as wells, channels, chambers, cavities, grooves, or recesses, which are designed to accommodate fluids, particles, cells, or other materials. In some embodiments, confinements may take the form of microstructures, such as capillaries, micropores, or microchambers.

[0058] Actuation source, as used herein, refers to a component that generates ultrasonic waves or vibrational energy, which is directed towards the resonance device. The actuation source may include piezoelectric transducers, electrostrictive devices, electromagnetic acoustic transducers, or other components capable of producing resonant actuation.

[0059] The present disclosure relates to an ultrasonic actuation device arranged for providing an ultrasonic actuation to at least one resonance structure of at least one resonance device / fluidic device. The ultrasonic actuation device may comprise a planar actuation frame arranged for accommodating the fluidic device in a fluidic device area. Additionally, the ultrasonic actuation device may comprise an actuation arrangement comprising at least one actuation source connected to the actuation frame. The actuation source may be connected to the actuation frame such that at least one actuation source does not overlap the fluidic device area, in a resonance direction of the actuation source. The presently disclosed ultrasonic actuation device may comprise an actuation arrangement, comprising or consisting of a plurality of actuation sources, such as one, two, three, and / or four actuation sources, such as to improve the actuation efficiency and / or to distribute heat across the actuation device. In one embodiment of the present disclosure, the ultrasonic actuation device comprises four actuation sources, leading to a distribution of the heat, and can negate the need for active cooling of the ultrasonic actuation device and / or the resonance structure(s).

[0060] One important reason to improve actuation efficiency is to limit the amount of heat generated. For example, within cell cultures, local heating and temperature control play an important role as elevated temperatures can affect cells negatively in different ways. Therefore, in one embodiment of the present disclosure, the ultrasonic actuation device comprises a plurality of actuation sources that are distributed over an area of the actuation frame, such as distributed over a face of the actuation frame.

[0061] In one of the embodiments of the presently disclosed ultrasonic actuation device, the actuation source is provided as a planar structure that is attached to the actuation frame, such as adhered, laminated and / or bonded to the actuation frame. The ultrasonic actuation device may be a planar structure comprising two opposing faces that are planar and parallel. Typically said two opposing faces are parallel with the general plane of the planar actuation frame. The actuation source(s) may further comprise three or more sidewalls connecting the opposing faces, such as wherein each of said sidewalls are perpendicular to the opposing faces. Typically, no pair of said sidewalls are parallel. Therefore, the actuation source may be arranged without parallel sidewalls. This may also be referred to as the actuation source having slanted cuts. By the above mentioned arrangement of the actuation source(s), lateral modes may be suppressed. In this way energy losses due to undesired vibrations can be prevented.

[0062] The quality factor (Q-factor) is an important parameter for the actuation source(s). The optimal values of the Q-factor depends on the application. The quality factor (Q-factor) for an actuation source, e.g. a piezoelectric, is a dimensionless parameter that characterizes the energy dissipation and efficiency of the actuation source. It is a measure of how well the actuation source can sustain oscillations at a resonant frequency. A higher Q-factor indicates lower energy losses and better energy transfer efficiency, but also implies a more narrow frequency operation range. The frequency operation range is characterized by the bandwidth (BW) and the Q-factor can be calculated using the BW and the resonance frequency fR as, Q = The optimal Cofactor depends on the application in the sense that a fluidic chip with multiple resonant structures, such as microwells, will require a wider bandwidth to cover all of their individual resonances. Thus, it is a preference that the Q-factor of the actuation source(s) is selected such that the resulting bandwidth covers all the individual resonances of the resonant structures.

[0063] In a preferred embodiment of the ultrasonic actuation device, the actuation source has a Q-factor in the range of between 10 and 1000, such as in the range between 20 and 500, ensuring efficient energy transfer and sustained oscillations. In addition, the Q- factor can be adapted to any desired frequency operation range for any microfluidic chip, by modifying the actuation sources. For example, one may select appropriate piezoelectric material, and / or modify the way that the actuation source(s) are connected, such as bonded, to the planar actuation frame (for instance using an adhesive).

[0064] In one embodiment of the present disclosure, the actuation source(s) may comprise or consist of a piezoelectric transducer, an electrostrictive device, and / or an electromagnetic acoustic transducer, or a combination thereof. Thus, the actuation arrangement may comprise or consist of one or more piezoelectric transducers, one or more electrostrictive devices, and / or one or more electromagnetic acoustic transducers, or a combination thereof.

[0065] In an embodiment of the present disclosure, the one or more actuation sources each consist of a piezoelectric transducer. The one or more piezoelectric transducers may be selected from materials such as crystalline piezoelectric materials, a polymeric piezoelectric material such as those made of PZT, BaTiO3, LiNbO3, AIN, ZnO, poly(vinylidene fluoride), and / or PVDF and / or lead-free materials. In the preferred embodiment the piezoelectric transducer is made of lead zirconium titanate (PZT).

[0066] In a preferred embodiment of the present disclosure, the one or more piezoelectric transducer is connected to the planar actuation frame. For example, the one or more piezoelectric transducers may be attached or mounted on the top face of the planar actuation frame, preferably positioned such that the one or more piezoelectric transducers are distributed over an area of the planar actuation frame. For example wherein each piezoelectric transducer is adjacent to a different edge of the fluidic device area. In one of the embodiments of the present disclosure, four piezoelectric transducers are connected to the actuation frame. The actuation sources may be arranged to distribute the generated heat through the actuation frame and / or provide a more uniform actuation of the resonance structure, such as the pressure fields of each resonance structure, by the use of a plurality of actuation sources.

[0067] Preferably, the resonance of the one or more actuation sources does not overlap with the fluidic device area. The one or more actuation sources may for example be arranged such that they do not overlap with the fluidic device area, in the resonance direction of the actuation source(s). For example, the device may be arranged such that no part of the one or more actuation sources overlap with the fluidic device area, such as in the resonance direction of the one or more actuation sources. In an embodiment of the present disclosure, the one or more actuation sources do not overlap the fluidic device area, such as in the plane of the planar actuation device.

[0068] The one or more actuation sources, such as the one or more piezoelectrics, may be arranged to generate vibrations at an orthogonal direction with respect to the plane of the planar actuation frame. At the same time, the one or more actuation sources may be arranged such that they do not overlap with the fluidic device area, in said orthogonal direction. This arrangement may be referred to herein as “lateral coupling”. Thus, the one or more actuation sources may be arranged such that they, in use, are laterally coupled to the fluidic device. The one or more actuation sources are typically arranged such that they, in use, are laterally coupled to any fluidic device that is received by the planar actuation frame, such as any device positioned at the fluidic device area.

[0069] The one or more actuation sources may have a polarization direction such that a projection of the actuation source, in the polarization direction onto the fluidic device plane, is outside of the fluidic device area.

[0070] In addition, in one embodiment of the presently disclosed ultrasonic actuation device, the planar actuation frame is arranged to be, during use, in a horizontal, and / or vertical, position. The general plane of the planar actuation frame may thereby be horizontal and / or vertical. Further, the polarization direction of the one or more actuation sources may be orthogonal to said plane of the planar actuation frame. Alternatively, the planar actuation frame may be arranged to be, during use, in a vertical position wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame.

[0071] As mentioned above, the actuation arrangement may be arranged such that the vibrations of the one or more actuation sources are, during use, provided to the one or more fluidic devices. For example by being laterally coupled to the one or more fluidic devices. The lateral coupling results in an increased design freedom, and ease of use. The lateral coupling configuration may allow for an ultrasonic actuation device / actuation frame that is adapted to accommodate and actuate multiple resonance devices / fluidic devices. The ultrasonic actuation device may comprise one or more actuation sources, which can be configured to operate at the same or at different frequencies, depending on the requirements of the application.

[0072] The present disclosure relates to an ultrasonic actuation device arranged for generating ultrasound and to actuate one or more fluidic devices. Typically, the fluidic devices are actuated in such a way that the main axial resonance does not include the one or more fluidic devices. In one embodiment of the present disclosure, the one or more fluidic devices are arranged to be received by a part of the actuation frame, such as positioned onto a part of said frame. Preferably, the device is arranged such that the one or more fluidic devices are to be received at or near the one or more fluidic device areas. For example the planar actuation frame may be arranged to receive the one or more fluidic devices by a holding arrangement, such as a lip or recess. Said holder arrangement may be adapted such that the one or more fluidic devices are retained in the fluidic device area and / or such that the one or more fluidic devices are retained in a position wherein they are laterally coupled to the one or more actuation sources.

[0073] The one or more fluidic devices may, for example, comprise one or more confinements, such as receptacles, for example one or more microwells. The one or more confinements, e.g. receptacles and / or microwells, may for example have at least a lower surface and side surfaces and may be integrated into a fluidic device. The lower surface of the confinements may for example be planar with the plane of the fluidic device and / or the plane of the planar actuation frame during use.

[0074] The one or more fluidic devices, and or the resonance structures of said fluidic devices, may be formed in any suitable substrate material, including silicon, glass, metals, or polymeric materials. Preferably the material of the fluidic devices and / or resonance structures are suitable for propagation of acoustic waves.

[0075] The fluidic devices may comprise multiple materials. For example the fluidic device may comprise one or more resonance structures that are in one or more different materials than the rest of the fluidic device. Preferably, the lower surfaces of the fluidic device, and / or the resonance structures, are formed of an optically transparent material, such as glass, thereby allowing for visual inspection, e.g. by optical microscopy, of the contents of the resonance structure(s), such as the microwell(s).

[0076] In the preferred embodiment, the fluidic devices may comprise glass such as a coverslip or glass slide bonded to a silicon structure to form at least one confinement arranged to contain particles and or cells.

[0077] In one embodiment of the presently disclosed ultrasonic actuation device, the fluidic devices contain either a single array with 100 wells (10 x10 array) or 6 x 6 wells each organized in 4 x 4 arrays. In one embodiment of the present disclosure, the fluidic devices may comprise a well plate, such as a multiwell plate, for example a 96-well plate.

[0078] In addition, the presently disclosed ultrasonic actuation device may comprise one or more actuation sources that are frequency matched to the planar actuation frame, in order to allow for a more efficient actuation, such as of the fluidic device.

[0079] The frequency matching between the one or more actuation sources and the planar actuation frame may for example be carried out by modifying the thickness of the actuation frame. In some examples, the tuning of the actuation frame may be achieved by decreasing the thickness of the actuation frame, such as by trimming. In one embodiment of the present disclosure, the planar actuation frame comprises or consists of a metal, such as aluminum. Thus, the planar actuation frame may be made of a metal, such as aluminum.

[0080] In one embodiment of the present disclosure, the actuation frame comprises an aperture, such as an opening. Preferably, the aperture is at least partly overlapping the fluidic device area. In this way, optical inspection of the fluidic device, such as by a microscope is enabled.

[0081] In one embodiment of the present disclosure, the fluidic device area is arranged with an unobstructed optical path across the fluidic device, such as perpendicular to the plane of the planar actuation frame, such as for all resonance structures of the fluidic device. The actuation frame may in this way be compatible with microscope setups, including microscope objectives having short working distances. In a preferred embodiment of the present disclosure, the actuation frame is arranged to be coupled to the fluidic device by a coupling agent, such as immersion oil, water, a synthetic oil, a semisynthetic oil, mineral oil, and / or a natural oil. The coupling agent may preferably be selected such that it allows for coupling between the fluidic device and the actuation frame. Further the coupling agent may be non-volatile liquid, such as an oil.

[0082] During use, the presently disclosed ultrasonic actuation device may be arranged such that it allows for simultaneous manipulation of suspended particles, and / or cells, in a plurality of discrete resonance structures, such as microwells, of the fluidic device. Particles and / or cells as used herein, refers to all types of minute entities.

[0083] Said minute entities may be living or dead, organic or inorganic, natural or synthetic, simple or complex, single particles or aggregates, or combinations thereof, having a size in the range of about 10 nm to about 1 mm.

[0084] Additionally, minute entities include but are not limited to, organic or inorganic particles, such as, functionalized or non-functionalized, polymer or metal particles, bubbles, droplets, prokaryotic cells such as bacteria or archaea, or eukaryotic cells, such as human or animal or plant cells, viruses, large molecules or molecular complexes, such as DNA molecules or antibodies. The term "manipulation", as used herein, generally refers to all kinds of controllable external influence on the particles and / or cells which cause a defined movement or holding of the particles / cells which would not occur without this external influence. Examples of such manipulation are merging, positioning, guiding, or separating particles or cells in a microwell of the fluidic device.

[0085] Particles and / or cells contained by the resonance structures are typically expected to align with one of the potential field lines as a result of the actuation of the resonance structures. The particular field line is determined by the space that they occupy, and therefore it will also be a result of the number and the size of the particles and / or cells. When handling cells for the purpose of generating multicellular constructs or organoids one may manipulate the shape by combining potential fields at different contributions. If using the fundamental resonance this would range from spherical to oval structures. Examples of such manipulation can be seen in Figure 8.

[0086] The presently disclosed ultrasonic actuation device has an improved power efficiency that is translated into improved throughput, flexibility, and lower cost, as well as improved uniformity of acoustic fields. The presently disclosed ultrasonic actuation device is preferably arranged such that resonance structures of a fluidic device are actuated more uniformly. This may be achieved by using the ultrasonic actuation device in the method as disclosed herein. Thus, by modifying the actuation sweep parameters in a unique way where each frequency is normalized to the efficiency of the source at that frequency, the resonance structures may be actuated more uniformly. For example, such that the potential fields of the resonance structures are more uniform. This is a significant advantage when actuating complex fluidic devices (e.g. fluidic devices with over 300 resonance structures).

[0087] In a further aspect, the present disclosure relates to a system comprising an ultrasonic actuation device as disclosed herein and a fluidic device.

[0088] In yet a further aspect, the present disclosure relates to a method for power- compensated calibration of an actuation arrangement comprising an actuation device. The method is not limited to any particular type of actuation device, for example, a laterally coupled actuation device, but is instead broadly applicable to various ultrasonic actuation systems with one or more actuation sources. These sources may be arranged in different spatial configurations relative to the fluidic device, including, but not limited to, lateral, vertical, or other alignments. The method may be particularly useful for systems where precise calibration of energy input across a range of frequencies is desired for effective actuation.

[0089] The present inventors have realized that while ultrasound frequency sweeps are typically able to produce homogenous cell aggregates when combined with broadband sources, in some configurations, using a matched actuator with a resonance peak in the sweep range can lead to uneven weighting of frequencies near the resonance peak. To mitigate this and provide the ability to utilize frequency sweeps in combination with efficient sources of acoustic energy, a strategy of power-compensated sweeps is proposed.

[0090] Thus, the presently disclosed method is directed at power-compensated calibration of an actuation arrangement comprising an actuation device. This method may be applied to a wide variety of actuation devices and configurations, including those where the actuation source is not laterally coupled to the fluidic device. The method can also be used in devices with actuation sources arranged in different spatial configurations, such as vertically aligned sources, sources distributed around a fluidic device, or other geometric configurations that involve multiple actuation sources or alternative resonance structures.

[0091] The presently disclosed method typically involves the step of measuring electrical impedance of the actuation device at a plurality of frequencies within a modulation bandwidth. The measured impedance information may be used to compensate for variations when sweeping the frequency, ensuring robust and uniform force fields across the fluidic device. The method is not restricted to a particular type of fluidic device or actuation source configuration, making it applicable to various devices with different coupling arrangements.

[0092] This flexibility ensures that the method can be used for any type of actuation device and actuation system. For example, the method may be used for actuation using actuation devices and / or actuation systems that differ from the laterally coupled systems as disclosed herein. For example actuation systems and devices that rely on using different actuation geometries or configurations, as well as systems with multiple actuation sources. For example, the method may be applied to actuation devices where the actuation source is positioned directly beneath or around the fluidic device, or in more complex fluidic systems with multiple resonance structures.

[0093] Thus, the presently disclosed method is directed at power-compensated calibration of an actuation arrangement comprising an actuation device. Typically, the actuation device comprises at least one actuation source arranged to be actuated at a constant voltage.

[0094] The method may comprise:

[0095] • measuring electrical impedance of the actuation device at a plurality of frequencies within a modulation bandwidth of the actuation device; and

[0096] • determining, for each frequency, based on the measured impedances, the time step needed to obtain an equal predetermined energy input.

[0097] The presently disclosed method allows for adaptive frequency modulation which can enable automatic calibration. This produces a tailored actuation scheme for each of a plurality of fluidic devices, which may subsequently be actuated by the actuation device, as shown in Figs. 6-9.

[0098] This method is typically based on analyzing the electrical impedance at each frequency within the modulation bandwidth. This information may be used to compensate for variations during frequency sweeping. This results in more robust and uniform force fields with similar magnitudes between different micro-wells or resonance structures compared with actuation methods of the prior art.

[0099] Among the advantages of this method is that fluidic devices can be inserted and removed from the actuating frame without a need for manual calibration of frequency and voltage before each experiment.

[0100] The presently disclosed method may be arranged such that it is assumed that the strength of each mode in the sweep is proportional to the energy input from the actuations source, En, during one sweep repetition, Here, Unis used to describe an intrinsic mode shape and amplitude that is determined by factors not directly related to excitation from the source, pn, is the active power delivered from the source, and tnis the time spent at that frequency during the sweep period.

[0101] To compensate for the presence of a resonance mode in the source and provide a homogenous excitation of all modes in the sweep, the time, tn, can be adjusted such that the energy input for each frequency is equal. Based on measurements of the electrical impedance the active power can be calculated as,

[0102] Pn = v2Gn. (2)

[0103] Where, v, is the applied voltage amplitude, and Gnis the conductance (real part of admittance) for the frequency in question. With this formula one can calculate the time step for each frequency needed to provide equal energy input to the other frequencies in the sweep.

[0104] The desired sweep power, p, and sweep period, T, and number of frequency steps, N, can then be used to equate the energy input of a single step to the average step energy input,

[0105] Pntn = P^, (3) resulting in,

[0106] This formula may be used to construct the power compensated sweep as an arbitrary waveform consisting of a plurality of frequencies. In some cases, the range of frequencies may include a resonance peak, such as one that is centered around a resonance peak. The arbitrary waveform can then be stored and generated by utilizing a standard function generator. Specifically, as shown in the example of Fig. 6, function (4) above was used to construct the power-compensated sweep as an arbitrary waveform consisting of 100 frequencies centered around 2.5 MHz with a 100 kHz range. The arbitrary waveform was stored and generated using a standard function generator.

[0107] Fig. 6 further shows the power supplied from the source at each frequency in the sweep range together with the time steps used to compensate for the various power deliveries.

[0108] The method may be implemented in various actuation systems using different types of amplifiers and electrical configurations. Although examples provided in the figures focus on specific configurations, such as those using a flat energy input for a wide frequency range, the method is not limited to these examples. The power-compensated calibration technique may be adapted to suit different actuation systems and sweep ranges. Whether the device is arranged with lateral coupling or other configurations, the method can still be used to provide an optimized and balanced energy input for each frequency step.

[0109] Additionally, while the method has been shown to work with specific fluidic devices and resonance structures, it is also compatible with other actuation setups beyond for example laterally coupled devices as described elsewhere herein. This includes, for example, systems with alternative resonance structures, such as vertically aligned actuation sources, multiwell plates, microfluidic devices, and other types of fluidic systems that require precise actuation control.

[0110] The method is not limited to the use of a specific type of amplifier. The presently disclosed method may be implemented by the use of other arrangements for actuation of the fluidic system and / or fluidic device. In some embodiments, the final voltage across the actuator is considered based on the actuator arrangement. For example, an instrument with a standard 50 Ohm output impedance may be used by including a calculation of voltage division for each step in the sweep.

[0111] The method may further be implemented as a computer-implemented method, wherein one or more steps of the method are executed by a processor or control system. The instructions for performing the method can be stored, fully or partly, on a computer- readable medium, including non-transitory memory or transient-memory, such as RAM, volatile memory, or other forms of temporary storage. The computer-implemented method may comprise control algorithms or software that perform measurements, calculations, and signal generation for the power-compensated calibration, ensuring the correct energy input across the frequency sweep. In some embodiments, the method may further comprise the analysis of additional data inputs, such as image data, to optimize the actuation parameters, allowing dynamic adjustments based on observed characteristics of the resonance device during operation.

[0112] In some embodiments, the control system may be integrated into the actuation arrangement or configured to communicate with external devices to facilitate real-time calibration and adjustments. This may include the use of data from imaging or other sensing technologies to further refine and control the actuation based on real-time feedback.

[0113] While the results shown in Figs. 6-8 have been obtained by the use of a flat energy input for a relatively wide sweep range, the presently disclosed method of power- compensated frequency sweeps may, of course, be modified and tailored to other circumstances.

[0114] Thus, using the same principle, other adjustments, such as modifications to timings or the application of different weights, may be applied to provide arbitrary mixing or balancing of effects occurring at various frequencies. In another embodiment, the sweep range may be modified to provide an optimal trade-off between power input and robustness. Such adjustments may serve as a means for tuning the bandwidth without making physical alterations to the device.

[0115] In a further aspect, the present disclosure relates to an ultrasonic actuation system for power-compensated calibration. The system may comprise an actuation arrangement having at least one actuation source, a control system configured to measure the electrical impedance of the actuation arrangement at a plurality of frequencies within a modulation bandwidth, and a processor configured to determine, for each frequency, the time step needed to obtain an equal predetermined energy input based on the measured impedances. The ultrasonic actuation system may further be configured to apply the method of power-compensated calibration as previously described, ensuring uniform energy input across the frequency sweep.

[0116] The control system of the ultrasonic actuation system may comprise software, hardware, or a combination thereof that is capable of executing the calibration method, including adjusting the energy input by calculating the impedance and adjusting the time steps accordingly. The control system may also be configured to generate a drive signal, based on the calculated time steps, to ensure the appropriate energy output at each frequency during the sweep.

[0117] In some embodiments, the ultrasonic actuation system may further comprise a current amplifier, such as one with a high output impedance, configured to apply a substantially constant voltage at each frequency step regardless of impedance variations across the actuation source. The system may also include a feedback mechanism that allows realtime adjustments to the energy input based on ongoing impedance measurements, ensuring precise control over the actuation process.

[0118] In another embodiment, the actuation arrangement may comprise multiple actuation sources, and the control system may be configured to independently control the energy input for each actuation source. This may allow the system to deliver tailored energy profiles to each actuation source, optimizing the acoustic environment for different regions of the fluidic device or multiple fluidic devices within the same system. The system may also be capable of handling varying geometries of fluidic devices, including multiwell plates, microfluidic channels, or other configurations, by independently controlling each actuation source’s energy output.

[0119] The ultrasonic actuation system may be implemented as part of a larger fluidic system where the fluidic device contains one or more resonance structures designed to interact with the ultrasonic waves generated by the actuation source. These resonance structures may include microwells, microchannels, or capillaries, which serve as confinements for the liquid, particles, or cells being manipulated during the actuation process. The control system may be configured to adjust the energy input across these different resonance structures to ensure uniform manipulation of the fluid or cells across the fluidic device. In some embodiments, the system may be controlled by a processor or control algorithm that performs the steps of measuring impedance, calculating time steps, and generating drive signals. The processor may be integrated into the system or communicate with external computing devices. The software or control algorithm may be stored on a computer-readable medium, which can include both non-transitory memory (such as flash memory or hard drives) and transient memory (such as RAM). This ensures flexibility in the system's ability to store and execute instructions, allowing for efficient real-time calibration and operation of the ultrasonic actuation system.

[0120] In yet another embodiment, the ultrasonic actuation system may be configured to be compatible with a variety of fluidic devices, including those that are not laterally coupled to the actuation source. The system can be adapted for various spatial configurations, including vertical, horizontal, or distributed arrangements of actuation sources. This flexibility ensures that the system can be used in a wide range of applications, from research laboratories to industrial settings, where precise ultrasonic actuation is required.

[0121] Detailed description of Drawings

[0122] The invention will in the following be described in greater detail with reference to the accompanying drawings. The drawings are exemplary and are intended to illustrate some of the features of the presently disclosed ultrasonic actuation device and related methods, and are not to be construed as limiting to the presently disclosed invention.

[0123] Fig. 1 shows a cross-section view of an ultrasonic actuation device (1) according to an embodiment of the present disclosure. The ultrasonic actuation device (1) is arranged for providing a reproducible and effective source of acoustic energy / ultrasonic actuation to a fluidic device (4). The actuation device (1) comprises a planar actuation frame (2), for example made of metal. The actuation device comprises an actuation arrangement comprising at least one actuation source (3), such as a number of piezoelectric transducers. The actuation sources are typically mounted on top of the planar actuation frame (2).

[0124] Further, the ultrasonic actuation device may comprise an aperture (6) for holding the fluidic device (4). The aperture (6) typically at least partly overlaps the fluidic device area. The planar actuation frame may comprise a holding arrangement (5), such as a recess or a lip, for example located adjacent to the aperture (6), such as around the perimeter of the aperture (6). The holding arrangement (5) is typically arranged such that the aperture (6) overlaps, at least partly, with the fluidic device area.

[0125] Fig. 2 shows a 3D schematic representation of the ultrasonic actuation device (1). The ultrasonic actuation device comprises a planar actuation frame (2). The planar actuation frame (2) comprises an aperture (6) that is arranged to overlap at least a part of the fluidic device area. Additionally, the planar actuation frame comprises a holding arrangement (5), that may be arranged to accommodate the fluidic device. In addition, Fig 2 shows an actuating source (3) attached to the top surface of the planar actuation frame (2).

[0126] Figs. 3A-B show a top view (Fig. 3A) and a 3D schematic representation (Fig. 3B) of the ultrasonic actuation device (1) showing a planar actuation frame (2) with four actuation sources (3’) that have been asymmetrically cut / slanted in order to optimize electrical properties. Thus, the actuations sources have non-parallel sidewalls. The planar actuation frame (2) accommodates a fluidic device (4) comprising a number of resonance structures. In this specific example, the resonance structures are a 4 x 4 array of separate wells / resonance structure (7) wherein each well or resonance structure has an array of 6 x 6 microwells (8). The configuration of the planar actuation frame (2) shown in this figure, provides optical access to the fluidic device (4) particularly for experimentation such as live imaging and high-content microscopy screening applications of 3D cell cultures.

[0127] Fig. 4A illustrates an embodiment of an ultrasonic actuation device (1). The ultrasonic actuation device (1) is arranged to provide acoustic energy to a fluidic device that can be accommodated by a planar actuation frame (2). In this embodiment, the frame (2) is designed with a square shape, contrasting with the rounded shape of the frame in Fig. 1 , and has a slightly reduced thickness. The actuation sources (3), such as piezoelectric transducers, are positioned on the top surface of the planar actuation frame (2) to generate ultrasonic vibrations. The ultrasonic actuation device (1) also includes an aperture (6) for allowing visual inspection of the fluidic device (4) from the opposite side of the actuation frame. The fluidic device may be accommodated at the fluidic device area, by a holding arrangement (5), such as a recess or lip, to secure the fluidic device in place. Fig. 4B shows an alternative embodiment of the ultrasonic actuation device (1), specifically adapted to accommodate multiple resonance devices or fluidic devices (4). In this example shown, the actuation frame (2) includes multiple fluidic device areas. In this example, each fluidic device area is associated with an individual aperture (6). The actuation arrangement may comprise one or more actuation sources (3), arranged to provide acoustic energy across the separate fluidic device areas or apertures, either at the same frequency or at varying frequencies depending on the requirements of the application. This setup allows for parallel operation and is suitable for high-throughput applications, enabling the independent or synchronized actuation of multiple resonance devices within a single ultrasonic actuation device (1).

[0128] Fig 5 shows admittance and susceptance spectra graphs for two different ultrasonic actuation device configurations, with resonances observable as peaks in the admittance graphs. Fig 5A shows an embodiment of the ultrasonic actuation device (1) wherein susceptance and admittance measurements results are obtained using a generic ultrasonic actuation device (1) with a planar actuation frame (2) as thick as 3mm and one piezoelectric transducer (3). The susceptance and admittance measurement results displayed a random set of 3 resonance peaks. Fig 4B shows an embodiment of the ultrasonic actuation device (1) wherein a KLM model is used to specify the planar actuation frame thickness (1.3 mm) needed to frequency match the main resonance peak with the operating frequency of the piezoelectric transducer (3), showing in this example as 2.5 MHz.

[0129] Fig 6 shows admittance and susceptance spectra graphs for different ultrasonic actuation device configurations, with resonances observable as peaks in the admittance graphs. Fig. 6A shows an embodiment of the ultrasonic actuation device (1), wherein the removal of unwanted resonance peaks is observed by utilizing nonparallel (asymmetrically / slanted) sidewalls of the actuation source (3’). Fig. 6B shows an embodiment of the ultrasonic actuation device wherein no significant impedance alteration of the ultrasonic actuation device’s spectrum is observed while holding a fluidic device (4) containing a single array with 100 wells (10 x10). The single resonance peak is maintained at around 2.5 Mhz. Fig. 6C shows an embodiment of the ultrasonic actuation device (1) wherein no significant impedance alteration of the ultrasonic actuation device’ spectra is observed while holding a fluidic device (4) containing 4 x 4 arrays / confinements with 6 x 6 wells each. The single resonance peak is still maintained at around 2.5 Mhz. Fig. 6B-C thus show that the presently disclosed actuation frame enables efficient and simple actuation of a large variation of fluidic devices, through having the actuation source(s) laterally coupled to the fluidic device(s).

[0130] Fig. 7 shows an arbitrary waveform for a power compensated sweep, consisting of 100 frequencies centered around 2.5 MHz with a 100 kHz range, that has been generated through the use of the presently disclosed method. The power supplied at each frequency (upper graph) is shown together with the time steps used to compensate for the various power deliveries (bottom graph). The active power delivered by the source is typically dependent on the electrical properties of the actuation source (3) and the applied voltage. If the same voltage is applied for all frequencies in a sweep the active power will mirror the shape of the admittance peak. To compensate, the time steps in the sweep can be adjusted such that equal energy is delivered to each frequency step in the sweep.

[0131] Fig. 8 shows a COMSOL Multiphysics simulation of pressure (p), velocity (v), and force potentials (II) (of a single II and combined U1+LI2). Simulations of the pressure and velocity fields produced at the fundamental resonance of a single well; this resonance consists of two orthogonal modes aligned diagonally in the well. Frequency sweeping can be used to combine the effect of multiple single frequencies into new composite fields. Sweeps are useful as they allow the formation of fields with new shapes, adds robustness to temperature fluctuations, and may allow dynamic manipulation and or stimulation of beads (e.g., cells). During a linear frequency sweep each angular frequency within the sweep, will generate a potential field which is averaged to generate a resulting potential. For example, in the present COMSOL simulation, the force potential is calculated for polystyrene beads showing how trapped beads are expected to align along the field lines. Depending on the space that the trapped beads occupy they can either form an ellipse (tilted) or a diagonal band during actuation. Additionally, Fig. 8 shows examples when the force potentials are combined, bottom row, (e.g., using a frequency sweep) with different weights. If one of the modes is dominant the trapped particles are expected to align in an elliptical aggregate, whereas if they are balanced a circular aggregate can be achieved. Fig. 9 shows an application of power compensation sweeps using beads. Additionally, it shows the quantification of the effect on beads’ aggregate shape and position in relation to a well within the fluidic device (4). Here, the difference between single frequency operation (A, D), linear sweeps (B, E), and power compensated sweeps (C, F) in terms of bead aggregation is shown.

[0132] In this particular example, 10 pm polystyrene beads are trapped and aggregated in a single resonance structure, such as a well, of a 10x10 well fluidic device (4). Fig. 9A, and D show the effects of a single frequency (SF), particularly at an actuation of 2.5 MHz, Fig. 9B, and E shows the effect of the linear sweep (LS) at 100 kHz at approximately 2.5 MHz, and Fig. 9C, and F show the effects of power compensated sweep (PCS) particularly at 100 kHz centered at approximately 2.5 MHz.

[0133] Fig. 10 shows a COMSOL Multiphysics simulation similar to that in Fig. 8, illustrating the distribution of pressure (p), velocity (v), and force potentials (II) within a confinement with a circular cross-sectional area. The resonance within this circular confinement exhibits radially symmetrical modes, resulting in similar trapping and alignment patterns as observed in Fig. 8. Frequency sweeping may be used to combine the effects of multiple single frequencies, creating composite fields that facilitate robust control and dynamic manipulation of particles within the circular confinement.

[0134] In this simulation, the force potential fields, calculated for polystyrene beads, indicate that trapped beads tend to align along the circular or radial field lines, ultimately forming central aggregates. Depending on the spatial distribution and combination of force potentials, the trapped beads may form various shapes, including circular or ringlike patterns. Fig. 11 shows the quantification of the aggregation of the particles shown in Fig. 9A-C by circularity (Fig. 11 A), minor axis to major axis ratio (Fig. 11 B), distance to the well center (Fig. 11 C), and area of the aggregates (Fig. 11 D). The results show that for the single frequency (SF) there was a large variation between the shapes (circularity) and positions of the aggregates in the well. Particularly the distance to the center of the wells and minor axis to major axis ratio. In a few instances, the beads were aligned in spherical aggregates in the center of the wells whereas the majority of the aggregates were elongated and sometimes attached to the sidewalls of the wells. In comparison to single-frequency actuation, applying the linear sweeps (LS) improved the uniformity of the bead aggregates providing more rounded shapes located closer to the center of the wells. However, when applying the power compensated sweep (PCSF) a further improvement of the shapes was observed in terms of circular aggregates minor axis to major axis to ratio, distance to the well center, and area of the beads aggregates.

[0135] Fig. 12 illustrates a flow chart of a method for power-compensated calibration of an ultrasonic actuation device, and / or a fluidic system, according to an embodiment of the present disclosure. This method enables precise calibration of the ultrasonic actuation arrangement by adjusting for variations in impedance across different frequencies, ensuring an even energy distribution across a frequency sweep. The method includes both essential and optional steps, as detailed below:

[0136] The method begins with the step of measuring electrical impedance of the actuation arrangement at a plurality of frequencies within the modulation bandwidth of the ultrasonic actuation device (121). This measurement enables identification of impedance characteristics across the frequency range and forms the basis for subsequent energy adjustments.

[0137] Based on the measured impedance values, time steps are determined (122) for each frequency to achieve an equal, predetermined energy input. This calculation allows the method to compensate for frequency-dependent variations in impedance, promoting a uniform excitation of the actuation source(s) across the frequency sweep.

[0138] In an optional step, the method may include generating a drive signal based on the calculated time steps (123). This drive signal may consist of a repetitive wave, incorporating each frequency and its corresponding time step, to ensure the desired energy output at each frequency in the sweep.

[0139] The method may further comprise a step of actuating at least one actuation source by applying the generated drive signal to the actuation arrangement (124). This optional step initiates the ultrasonic actuation of the fluidic system or device, enabling precise manipulation of the energy input across the range of frequencies. This actuation step may for example be performed at a substantially constant voltage, or at a voltage that varies minimally (e.g., less than 10%, such as less than 5%) to maintain consistent energy input.

[0140] This method can be configured for actuation devices with single or multiple actuation sources, and may include the use of a current amplifier with high output impedance to stabilize the applied voltage across frequency variations. In certain embodiments, this method may be computer-implemented, with control algorithms or software stored on a computer-readable medium, facilitating real-time calibration and adjustments.

[0141] Figure Reference list:

[0142] 1 Ultrasonic actuation device

[0143] 2 Actuation frame

[0144] 3 Actuation source

[0145] 4 Fluidic device

[0146] 5 Holding arrangement

[0147] 6 Fluidic device area

[0148] 7 Well / confinement

[0149] 8 Resonance structures

[0150] 121 measuring

[0151] 122 determining

[0152] 123 generating

[0153] 124 actuation

[0154] Items

[0155] 1. An ultrasonic actuation device arranged for providing an ultrasonic actuation to at least one resonance structure of at least one fluidic device, the actuation device comprising:

[0156] • a planar actuation frame comprising a fluidic device area arranged for accommodating the fluidic device; and

[0157] • an ultrasonic actuation arrangement comprising at least one actuation source connected to the actuation frame, wherein the at least one actuation source does not overlap the fluidic device area, in a resonance direction of the actuation source.

[0158] 2. The ultrasonic actuation device according to item 1 , wherein the actuation arrangement comprises a plurality of actuation sources, such as one , two, three, or four.

[0159] 3. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source is one or more piezoelectric transducers, electrostrictive devices, and / or electromagnetic acoustic transducers, or a combination thereof.

[0160] 4. The ultrasonic actuation device according to any one of items 2-3, wherein the actuation sources are positioned such that they are distributed over an area of the planar actuation frame, such as distributed over a face of the planar actuation frame.

[0161] 5. The ultrasonic actuation device according to any one of items 2-4, wherein the actuation sources are arranged such that each actuation source is adjacent to a different edge of the fluidic device area.

[0162] 6. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source is attached, such as adhered, to the planar actuation frame.

[0163] 7. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source comprises or consists of a piezoelectric material. 8. The ultrasonic actuation device according to item 7, wherein the piezoelectric material is selected from a crystalline piezoelectric material, a ceramic piezoelectric material, or a polymeric piezoelectric material, such as wherein the piezoelectric material is PZT, BaTiO3, LiNbOa, AIN, ZnO, poly(vinylidene fluoride), and / or PVDF.

[0164] 9. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source comprises or consists of a lead free material.

[0165] 10. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source does not overlap the fluidic device area, such as in the plane of the planar actuation device.

[0166] 11 . The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source comprises one or more planar structures, such as laminated to the planar actuation frame.

[0167] 12. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source comprises two planar and parallel opposing faces connected by three or more sidewalls, and wherein none of said sidewalls are parallel.

[0168] 13. The ultrasonic actuation device according to any one of the preceding items, wherein the fluidic device area is arranged in the plane of the planar actuation frame.

[0169] 14. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation source has a polarization direction such that a projection of the actuation source, in the polarization direction onto a plane of the fluidic device area, and / or the fluidic device, is outside of the fluidic device area.

[0170] 15. The ultrasonic actuation device according to any one of the preceding items, wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame.

[0171] 16. The ultrasonic actuation device according to any one of the preceding items, wherein the planar actuation frame is arranged to be, during use, horizontal and wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame. 17. The ultrasonic actuation device according to any one of the preceding items, wherein the planar actuation frame is arranged to be, during use, vertical and wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame.

[0172] 18. The ultrasonic actuation device according to any one of the preceding items, wherein the ultrasonic actuation device is arranged to accommodate multiple fluidic devices, and wherein each of said fluidic devices comprises at least one resonance structure.

[0173] 19. The ultrasonic actuation device according to any one of the preceding items, wherein the fluidic device is arranged to be placed onto a part of the actuation frame.

[0174] 20. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation frame comprises a means of accommodating the fluidic device, such as a recess or a lip.

[0175] 21. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation frame is arranged to be coupled to the fluidic device by a coupling agent, such as immersion oil.

[0176] 22. The ultrasonic actuation device according to any one of the preceding items, wherein the fluidic device comprises a cover slip, a glass slide, and / or a well plate, such as a 96 well plate.

[0177] 23. The ultrasonic actuation device according to any one of the preceding items, wherein the fluidic device comprises a planar glass structure, such as a cover slip or a glass slide, that defines the at least one resonance structure.

[0178] 24. The ultrasonic actuation device according to any one of the preceding items, wherein the at least one resonance structure is arranged for containing particles and / or cells, such as wherein the resonance structure is a confinement.

[0179] 25. The ultrasonic actuation device according to any one of the preceding items, wherein the at least one resonance structure is a well, such as a microwell. 26. The ultrasonic actuation device according to any one of the preceding items, wherein the fluidic device is a multiwell plate, a microfluidic device, a capillary device.

[0180] 27. The ultrasonic actuation device according to any one of the preceding items, wherein the fluidic device comprises or consists of glass, a polymer and / or a metal.

[0181] 28. The ultrasonic actuation device according to any one of the preceding items, wherein the actuation frame comprises an aperture that is arranged to overlap at least a part of the fluidic device area.

[0182] 29. The ultrasonic actuation device according to item 28, wherein the actuation frame comprises an aperture that is arranged to overlap at least a part of the fluidic device area; and wherein the actuation frame comprises a means of accommodating the fluidic device, arranged towards the fluidic device area.

[0183] 30. The ultrasonic actuation device according to any one of the preceding items, wherein the thickness of the planar actuation frame is selected such that the planar actuation frame is frequency matched with the resonance frequency of the at least one actuation source.

[0184] 31. The ultrasonic actuation device according to any one of the preceding items, wherein the planar actuation frame is made of metal, such as aluminum or copper, ceramic, polymer, and / or a composite material.

[0185] 32. The ultrasonic actuation device according to any one of the preceding items, wherein the device is arranged to be accommodated by a microscope stage, an incubator, and / or stage climate systems.

[0186] 33. The ultrasonic actuation device according to any one of the preceding items, wherein the device is arranged such that the fluidic device can be observed in a microscope, such as a compound microscope.

[0187] 34. The ultrasonic actuation device according to any one of the preceding items, wherein the device is arranged such that the resulting shape of the acoustic field and / or the magnitude of the acoustic field is similar, and / or identical, in a plurality of resonance structures of the at least one fluidic device. 35. The ultrasonic actuation device according to any one of the preceding items, wherein the device is arranged for producing at least one 3D cell culture, such as spheroid(s) and / or organoid(s) in the at least one resonance structure of the at least one fluidic device.

[0188] 36. The ultrasonic actuation device according to any one of the preceding items, wherein the device relies on passive cooling, and wherein the temperature during use remains within normal culturing conditions for the cells.

[0189] 37. The ultrasonic actuation device according to any one of the preceding items, wherein the device is arranged such that the fluidic device can be replaced by another fluidic device, such that the device can be reused multiple times with different fluidic devices.

[0190] 38. A fluidic system comprising the ultrasonic actuation device according to any one of the preceding items and a fluidic device.

[0191] 39. The system according to item 38, wherein the system is arranged for growth of 3D cell cultures, such as in wells of the fluidic device.

[0192] 40. Use of the system according to any one of items 38-39 for production of 3D cell cultures, such as in wells of the fluidic device.

[0193] 41. A method for power-compensated calibration of an ultrasonic actuation device, and / or a fluidic system, comprising an ultrasonic actuation arrangement having at least one actuation source, the method comprising:

[0194] • measuring electrical impedance of the actuation arrangement at a plurality of frequencies within a modulation bandwidth of the actuation device;

[0195] • determining, for each frequency, based on the measured impedances, the time step needed to obtain an equal predetermined energy input.

[0196] 42. The method according to item 41 , wherein the method comprises generating a drive signal, based on the time steps. 43. The method according to item 42, wherein the drive signal comprises a wave, such as a repetitive wave, that comprises each frequency during the respective time step, such that the predetermined energy output is obtained for each frequency.

[0197] 44. The method according to any one of items 41-43, wherein the fluidic system is arranged according to any one of items 38-39.

[0198] 45. The method according to any one of items 41-44, wherein the method comprises actuation of the at least one actuation source, by providing the drive signal to the actuation source.

[0199] 46. The method according to any one of items 41-45, wherein the actuation comprises actuation at a substantially constant voltage and / or wherein the drive signal is provided to the actuation arrangement at a substantially constant voltage, such as at a constant voltage and / or a voltage that varies less than 10 %, such as less than 5 %.

[0200] 47. The method according to any one of items 41-46, wherein the actuation arrangement comprises multiple actuation sources.

[0201] 48. A fluidic assembly comprising the fluidic system according to any one of items 38- 39, wherein the fluidic system is arranged for power-compensated calibration by the method of any one of items 41-47.

Claims

Claims1. An ultrasonic actuation device arranged for providing an ultrasonic actuation to at least one resonance structure of at least one fluidic device, the actuation device comprising:• a planar actuation frame comprising one or more fluidic device areas arranged for accommodating one or more fluidic devices; and• an ultrasonic actuation arrangement comprising at least one actuation source connected to the actuation frame, wherein the at least one actuation source does not overlap the fluidic device area, in a resonance direction of the actuation source.

2. The ultrasonic actuation device according to claim 1 , wherein the actuation arrangement comprises a plurality of actuation sources, such as one, two, three, or four.

3. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source is one or more piezoelectric transducers, electrostrictive devices, electroacoustic transducers, magnetostrictive devices, and / or electromagnetic acoustic transducers, or a combination thereof.

4. The ultrasonic actuation device according to any one of claims 2-3, wherein the actuation sources are positioned such that they are distributed over an area of the planar actuation frame, such as distributed over a face of the planar actuation frame.

5. The ultrasonic actuation device according to any one of claims 2-4, wherein the actuation sources are arranged such that each actuation source is adjacent to a different edge of the fluidic device area.

6. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source is attached, such as adhered, to the planar actuation frame.

7. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source comprises or consists of a piezoelectric material.

8. The ultrasonic actuation device according to claim 7, wherein the piezoelectric material is selected from a crystalline piezoelectric material, a ceramic piezoelectric material, or a polymeric piezoelectric material, such as wherein the piezoelectric material is PZT, BaTiO3, LiNbOa, AIN, ZnO, poly(vinylidene fluoride), and / or PVDF.

9. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source comprises or consists of a lead free material.

10. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source does not overlap the fluidic device area, such as in the plane of the planar actuation device.11 . The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source comprises one or more planar structures, such as laminated to the planar actuation frame.

12. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source comprises two planar and parallel opposing faces connected by three or more sidewalls, and wherein none of said sidewalls are parallel.

13. The ultrasonic actuation device according to any one of the preceding claims, wherein the fluidic device area is arranged in the plane of the planar actuation frame.

14. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation source has a polarization direction such that a projection of the actuation source, in the polarization direction onto a plane of the fluidic device area, and / or the fluidic device, is outside of the fluidic device area.

15. The ultrasonic actuation device according to any one of the preceding claims, wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame.

16. The ultrasonic actuation device according to any one of the preceding claims, wherein the planar actuation frame is arranged to be, during use, horizontal and wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame.

17. The ultrasonic actuation device according to any one of the preceding claims, wherein the planar actuation frame is arranged to be, during use, vertical and wherein the polarization direction of the actuation source is orthogonal to the plane of the planar actuation frame.

18. The ultrasonic actuation device according to any one of the preceding claims, wherein the ultrasonic actuation device is arranged to accommodate and actuate multiple fluidic devices simultaneously, and wherein each of said fluidic devices comprises at least one resonance structure.

19. The ultrasonic actuation device according to any one of the preceding claims, wherein the fluidic device is arranged to be placed onto a part of the actuation frame.

20. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation frame comprises a means of accommodating the fluidic device, such as a recess or a lip.

21. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation frame is arranged to be coupled to the fluidic device by a coupling agent, such as immersion oil.

22. The ultrasonic actuation device according to any one of the preceding claims, wherein the fluidic device comprises a cover slip, a glass slide, and / or a well plate, such as a 96 well plate.

23. The ultrasonic actuation device according to any one of the preceding claims, wherein the fluidic device comprises a planar glass structure, such as a cover slip or a glass slide, that defines the at least one resonance structure.

24. The ultrasonic actuation device according to any one of the preceding claims, wherein the at least one resonance structure is arranged for containing particles and / or cells, such as wherein the resonance structure is a confinement.

25. The ultrasonic actuation device according to any one of the preceding claims, wherein the at least one resonance structure is a well, such as a microwell.

26. The ultrasonic actuation device according to any one of the preceding claims, wherein the fluidic device is a multiwell plate, a microfluidic device, a microwell array, and / or a capillary device.

27. The ultrasonic actuation device according to any one of the preceding claims, wherein the fluidic device comprises or consists of glass, a polymer and / or a metal.

28. The ultrasonic actuation device according to any one of the preceding claims, wherein the actuation frame comprises an aperture that is arranged to overlap at least a part of the fluidic device area.

29. The ultrasonic actuation device according to claim 28, wherein the actuation frame comprises an aperture that is arranged to overlap at least a part of the fluidic device area; and wherein the actuation frame comprises a means of accommodating the fluidic device, arranged towards the fluidic device area.

30. The ultrasonic actuation device according to any one of the preceding claims, wherein said device comprises multiple fluidic device areas, wherein each of said multiple fluidic device areas is arranged for receiving a separate fluidic device.

31. The ultrasonic actuation device according to any one of the preceding claims, wherein the thickness of the planar actuation frame is selected such that the planar actuation frame is frequency matched with the resonance frequency of the at least one actuation source.

32. The ultrasonic actuation device according to any one of the preceding claims, wherein the planar actuation frame is made of metal, such as aluminum or copper, ceramic, polymer, and / or a composite material.

33. The ultrasonic actuation device according to any one of the preceding claims, wherein the device is arranged to be accommodated by a microscope stage, an incubator, and / or stage climate systems.

34. The ultrasonic actuation device according to any one of the preceding claims, wherein the device is arranged such that the fluidic device can be observed in a microscope, such as a compound microscope.

35. The ultrasonic actuation device according to any one of the preceding claims, wherein the device is arranged such that the resulting shape of the acoustic field and / or the magnitude of the acoustic field is similar, and / or identical, in a plurality of resonance structures of the at least one fluidic device.

36. The ultrasonic actuation device according to any one of the preceding claims, wherein the device is arranged for producing at least one 3D cell culture, such as spheroid(s) and / or organoid(s) in the at least one resonance structure of the at least one fluidic device.

37. The ultrasonic actuation device according to any one of the preceding claims, wherein the device relies on passive cooling, such as wherein the temperature during use remains within normal culturing conditions for the cells.

38. The ultrasonic actuation device according to any one of the preceding claims, wherein the device is arranged such that the fluidic device can be replaced by another fluidic device, such that the device can be reused multiple times with different fluidic devices.

39. A fluidic system comprising the ultrasonic actuation device according to any one of the preceding claims and a fluidic device.

40. The system according to claim 39, wherein the system is arranged for growth of 3D cell cultures, such as in wells of the fluidic device.

41. Use of the system according to any one of claims 39-40 for production of 3D cell cultures, such as in wells of the fluidic device.

42. A method for power-compensated calibration of an ultrasonic actuation device, and / or a fluidic system, comprising an ultrasonic actuation arrangement having at least one actuation source, the method comprising:• measuring electrical impedance of the actuation arrangement at a plurality of frequencies within a modulation bandwidth of the actuation device;• determining, for each frequency, based on the measured impedances, the time step needed to obtain an equal predetermined energy input.

43. The method according to claim 42, wherein the method comprises generating a drive signal, based on the time steps.

44. The method according to claim 43, wherein the drive signal comprises a wave, such as a repetitive wave, that comprises each frequency during the respective time step, such that the predetermined energy output is obtained for each frequency.

45. The method according to any one of claims 42-44, wherein the fluidic system is arranged according to any one of claims 39-40.

46. The method according to any one of claims 42-45, wherein the method comprises actuation of the at least one actuation source, by providing the drive signal to the actuation source.

47. The method according to any one of claims 42-46, wherein the actuation comprises actuation at a substantially constant voltage and / or wherein the drive signal is provided to the actuation arrangement at a substantially constant voltage, such as at a constant voltage and / or a voltage that varies less than 10 %, such as less than 5 %.

48. The method according to any one of claims 42-47, wherein the actuation arrangement comprises multiple actuation sources.

49. The method according to any one of claims 42-48, wherein the time steps are adjusted to compensate for variations in energy input across a range of frequencies, ensuring a homogenous excitation of all modes in the frequency sweep.

50. The method according to any one of claims 42-49, wherein the method is configured for use with actuation devices with non-uniform impedance characteristics, and the method compensates for such variations during the frequency sweep.

51. The method according to any one of claims 42-50, wherein the actuation arrangement further comprises a current amplifier, such as one with a high output impedance, to ensure substantially the same voltage is applied at each frequency step regardless of impedance variations.

52. The method according to any one of claims 42-51 , wherein the method is a computer-implemented method.

53. A fluidic assembly comprising the fluidic system according to any one of claims 39- 40, wherein the fluidic system is arranged for power-compensated calibration by the method of any one of claims 42-52.

54. An ultrasonic actuation system for power-compensated calibration, comprising:• an actuation arrangement having at least one actuation source;• a control system configured to measure the electrical impedance of the actuation arrangement at a plurality of frequencies within a modulation bandwidth; and• a processor configured to determine, for each frequency, the time step needed to obtain an equal predetermined energy input based on the measured impedances.

55. The ultrasonic actuation system according to claim 54, further comprising a current amplifier with a high output impedance, configured to apply a substantially constant voltage at each frequency step.

56. The ultrasonic actuation system according to any one of claims 54-55, wherein the actuation arrangement comprises multiple actuation sources, and the control system is configured to independently control the energy input for each actuation source.

57. The ultrasonic actuation system according to any one of claims 54-56, wherein the system is arranged to carry out the method of any one of claims 42-52.

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